WO1998045452A2

WO1998045452A2 - Enzyme derived from thermophilic organisms that functions as a chromosomal replicase, and preparation and uses thereof

Info

Publication number: WO1998045452A2
Application number: PCT/US1998/006921
Authority: WO
Inventors: Olga Yurieva; John Kuriyan; Michael E. O'donnell; David Jeruzalmi
Original assignee: The Rockfeller University
Priority date: 1997-04-08
Filing date: 1998-04-08
Publication date: 1998-10-15
Also published as: EP0983365A1; WO1998045452A3; AU7683998A

Abstract

A DNA Polymerase has been identified in a thermophile that functions as a chromosomal replicase. The specific enzyme is a holoenzyme III that has been identified in Thermus thermophilus, and corresponds to Polymerase III in E. coli. The genes and the polypeptides corresponding to T.th.. η, τ, ε, α and β subunits that they encode are disclosed, as are probes, vectors, methods of preparation and the methods of use. The enzymes of the present invention and their components are particularly well suited for use in procedures for the preparation of DNA, such as PCR, because of the speed and accuracy that they are able to achieve.

Description

ENZYME DERIVED FROM THERMOPHILIC ORGANISMS THAT FUNCTIONS AS A CHROMOSOMAL REPLICASE, AND PREPARATION

AND USES THEREOF

FIELD OF THE INVENTION

The present invention relates to thermostable DNA polymerases, and more particularly to such polymerases as can serve as chromosomal replicases and are derived from thermophilic bacteria. More particularly, the invention extends to DNA polymerase Ill-type enzymes from thermophilic bacteria, including recombinant subunits thereof, to isolated DNA coding for such polymerases which hybridizes to DNA probes prepared from the DNA sequence coding for T. thermophilus and its subunits, to DNA and antibody probes employed in isolation of said DNA, as well as to related methods for isolating said DNA and methods to express and purify the DNA and its subunits from the respective genes such as dnaX, dnaA, dnaN. dnaQ, dnaE and the like. The invention also relates to the purification and use of T. thermophilus^'? 1 Ill-type enzymes in efficient replication of a long natural template.

BACKGROUND OF THE INVENTION

Thermostable DNA polymerases have been disclosed previously as set forth in U.S. Patent No. 5,192.674 to Oshima et al., U.S. Patent Nos. 5,322,785 and 5,352.778 to Comb et al., and U.S. Patent No. 5,545,552, and others. All of the noted references recite the use of polymerases as important catalytic tools in the practice of molecular cloning techniques such as polymerase chain reaction (PCR). Each of the references states that a drawback of the extant polymerases are their limited thermostability, and consequent useful life in the participation in PCR. Such limitations also manifest themselves in the inability to obtain extended lengths of nucleotides, and in the instance of Taq polymerase, the lack of 3' to 5' exonuclease activity, and the drawback of the inability to excise misinserted nucleotides (Tindall, et al. (1990) Biochemistry 29:5226-5231). More generally, such polymerases, including those disclosed in the referenced patents, are of the Polymerase I variety as they have are approximately 90-95kDa in size and may have 5' to 3' exonuclease activity. They define a single subunit with concomitant limits on their ability to hasten the amplification process and to promote the rapid preparation of longer strands of DNA.

Chromosomal replicases are composed of several subunits in all organisms (Kornberg and Baker, 1992). In keeping with the need to replicate long chromosomes, replicases are rapid and highly processive multiprotein machines. All cellular replicases examined to date derive their processivity from one subunit that is shaped like a ring and completely encircles DNA (Kuriyan and O'Donnell, 1993 ; Kelman and O'Donnell. 1994). This "sliding clamp" subunit acts as a mobile tether for the polymerase machine (Stukenberg et. al., 1991). The sliding clamp does not assemble onto the DNA by itself, but requires a complex of several proteins, called a "clamp loader" which couples ATP hydrolysis to the assembly of sliding clamps onto DNA (O'Donnell et. al.. 1992). Hence, cellular replicases are classically comprised of three components: a clamp, a clamp loader, and the DNA polymerase. and for purposes of the present invention, the foregoing components also serve as a broad definition of a "Pol Ill-type enzyme".

DNA polymerase III holoenzyme (Pol III holoenzyme) is the multi-subunit replicase of the E. coli chromosome. Pol III holoenzyme is distinguished from Pol I type DNA polymerases by its high processivity (>50 kbp) and rapid rate of synthesis (750 nts/s) (reviewed in Kornberg and Baker, 1991; Kelman and O'Donnell, 1995). The high processivity and speed is rooted in a ring shaped subunit, called β, that encircles DNA and slides along it while tethering the Pol III holoenzyme to the template (Stukenberg et. al., 1991 ; Kong et. al., 1992). The ring shaped β clamp is assembled around DNA by the multisubunit clamp loader, called γ complex. The γ complex couples the energy of ATP hydrolysis to the assembly of the β clamp onto DNA. This γ complex clamp loader is an integral component of the Pol III holoenzyme particle. A brief -

overview of the organization of subunits within the holoenzyme and their function follows.

Pol III holoenzyme consists of 10 different subunits. some of which are present in multiple copies for a total of 18 polypeptide chains (Onrust et. al., 1995b). The organization of these subunits in the holoenzyme particle is illustrated in Fig. 1. As depicted in the diagram, the subunits of the holoenzyme can be grouped functionally into three components: 1) the DNA polymerase III core is the catalytic unit and consists of the α (DNA polymerase), e (3'-5' exonuclease) and θ subunits (McHenry and Crow, 1979), 2) the β "sliding clamp" is the ring shaped protein that secures the core polymerase to DNA for processivity (Kong et. al., 1992), and 3) the 5 protein γ complex (γδδ'χψ) is the "clamp loader" that couples ATP hydrolysis to assembly of β clamps around DNA (O'Donnell, 1987; Maki and Kornberg, 1988). A dimer of the τ subunit acts as a "macromolecular organizer" holding together two molecules of core and one molecule of γ complex forming the Pol IIP" subassembly (Onrust et. al., 1995b). This organizing role of τ to form Pol III* is indicated in the center of Fig. 1. Two β dimers associate with the two cores within Pol III* to form the holoenzyme capable of replicating both strands of duplex DNA simultaneously (Maki et. al.. 1998).

The DNA polymerase III holoenzyme assembles onto a primed template in two distinct steps. In the first step, the γ complex assembles the β clamp onto the DNA. The γ complex and the core polymerase utilize the same surface of the β ring and they cannot both utilize it at the same time (Naktinis et. al., 1996). Hence, in the second step the γ complex moves away from β thus allowing access of the core polymerase to the β clamp for processive DNA synthesis. The γ complex and core remain attached to each other during this switching process by the τ subunit organizer.

The γ complex consists of 5 different subunits

An overview of the mechanism of the clamp loading process follows. The δ subunit is the major touch point to the β clamp and leads to ring opening, but δ is buried within γ complex such that contact with β is prevented (Naktinis et. al., 1995). The γ subunit is the ATP interactive protein but is not an ATPase by itself (Tsuchihashi and Kornberg, 1989). The δ' subunit bridges the δ and γ subunits resulting in a γδδ' complex that exhibits DNA dependent ATPase activity and is competent to assemble clamps on DNA

(Onrust et. al., 1991 ). Upon binding of ATP to γ, a change in the conformation of the complex exposes δ for interaction with β (Naktinis et. al., 1995). The function of the smaller subunits, χ and ψ, is to contact SSB (through χ) thus promoting clamp assembly and high processivity during replication (Kelman and O'Donnell, 1995).

The three component Pol Ill-type enzyme in eukaryotes contains a clamp that has the same shape as E. coli β, but instead of a homodimer it is a heterotrimer. This hetertrimeric ring, called PCNA (proliferating cell nuclear antigen), has 6 domains like β. but instead of each PCNA monomer being composed of 3 domains and dimerizing to form a 6 domain ring {e.g. like β), the PCNA monomer has 2 domains and it trimerizes to form a 6 domain ring (Krishna et. al, 1994; Kuriyan and

O'Donnell, 1993). The chain fold of the domains are the same in prokaryotes (β) and eukaryotes (PCNA) and thus the rings have the same overall 6-domain ring shape. The clamp loader of the eukaryotic Pol Ill-type replicase is called RFC (Replication factor C) and it consists of subunits having homolgy to the γ and δ' subunits of the E. coli γ complex. The eukaryotic DNA polymerase Ill-type enzyme contains either of two DNA polymerases, DNA polymerase δ and DNA polymerase e. It is entirely conceivable that yet other types of DNA polymerases can function with either a PCNA or β clamp to form a Pol Ill-type enzyme (for example, DNA polymerase II of E. coli functions with the β subunit placed onto DNA by the γ complex clamp loader). The bacteriophage T4 also utilizes a Pol Ill-type 3 -component replicase. The clamp is a homotrimer like PCNA, called gene 45 protein. The gene 45 protein forms the same 6-domain ring structure as β and PCNA. The clamp loader is a complex of two subunits called the gene 44/62 protein complex. The DNA polymerase is the gene 43 protein and it is stimulated by the gene 45 sliding clamp when it is assembled onto DNA by the 44/62 protein clamp loader. The Pol Ill-type enzyme may be either bound together into one particle (e.g., E. coli Pol III holoenzyme), or its three components may not be assembled together into a stable particle in solution (like the eukaryotic Pol Ill-type replicases).

There is an early report on separation of three DNA polymerases from T.th. cells, however each polymerase form was reminiscent of the preexisting types of DNA polymerase isolated from thermophiles in that each polymerase was in the 110,000- 120,000 range and lacked 3'-5' exonuclease activity (Ruttimann et. Al.. 1985). These are well below the molecular weight of Pol Ill-type complexes that contain in addition to the DNA polymerase subunit, other subunits such as gamma and tau. Although the three polymerases displayed some differences in activity (column elution behavior, and optimum divalent cation, template, and temperatures) it seems likely that these three forms were either different repair type polymerases or derivatives of one repair enzyme (e.g. Pol I) that was modified into three forms by post translational modification(s) that altered their properties (e.g. phosphorylation, methylation. slight proteolytic clipping of residues that alter activity, or association with different ligands such as a small protein or contaminating DNA). Despite this previous work, it remained to be demonstrated that thermophiles harbor a Pol Ill-type enzyme that contain multiple subunits such as gamma and/or tau, functioned with a sliding clamp accessory protein, or could extend a primer over a long stretch of ssDNA.

Ruttimann, C, Cotoras, M., Zaldivar, J., and Vicuna, R. (1986) DNA polymerases from the extremely thermophilic bacterium Termus thermophilus HB-8. European J., of Biochem. 149, 41-46.

Previously it was not known how thermophilic bacteria replicated - only Pol I's have been reported. By distinction, chromsomal replicases such as Polymerase III identified in E. coli. if available in a thermostable bacterium, with all its accessory subunits, could provide a great improvement over the Polymerase I's, in that they are generally much more efficient - about 5 times faster and much more highly processive. Hence, one may expect faster and longer chain production in PCR. and higher quality of DNA sequencing ladders. Clearly the ability to practice such synthetic techniques as PCR would be enhanced by these methods disclosed for how to obtain genes and subunits of DNA polymerase III holoenzyme from thermophilic sources.

SUMMARY OF THE INVENTION

In accordance with the present invention, DNA Polymerase Ill-type enzymes as defined herein are disclosed that may be isolated and purified from a thermophilic bacterial source, that can function as a chromosomal replicase, and that possesses all of the structural and processive advantages sought and recited above. More particularly, the invention extends to the Polymerase Ill-type enzymes derived from thermostable thermophilic bacteria that exhibit the ability to extend a primer over a long stretch of ssDNA at elevated temperature, the ability to be stimulated by a cognate sliding clamp of the type that is assembled on DNA by a 'clamp' loader (e.g. γ complex), have clamp loading sub-units that show DNA stimulated ATPase activity at elevated temperature and/or ionic strength, and have a DNA polymerase-associated 3'-5' exonuclease activity (e.g., e subunit). Representative thermophiles include polymerases isolated from the thermophilic bacteria Thermus thermophilus (T.th. polymerase), Thermococcus litoralis (77/ or VENT ^I polymerase), Pyrococcus furiosus (Pfu or DEEPVENT polymerase), Pyrococcus woosii (Pwo polymerase) and other Pyrococcus species, Bacillus ster other mophilus (Bst polymerase), sulfolobus acidocaldarius (Sac polymerase), thermoplasma acidophilum (Tac polymerase), Thermus favus (Tfl/Tub polymerase), Thermus ruber (Tru polymerase), Thermus brockianus (DYNAZYME™ polymerase). Thermotoga neapolitana (Tne polymerase; See WO 96/10640). Thermotoga maritima (Tma polymerase; See U.S. Patent No. 5,374,553) and other species of the Thermotoga genus (Tsp polymerase) and Methanobacterium thermoautotrophicum (Mth polymerase). In a preferred embodiment, the thermophilic comprise those of the thermus and thermotoga species, and particularly T.th. And Tne and Tma.

A particular Polymerase Ill-type enzyme in accordance with the invention may include at least one of the following sub-units: A. a γ subunit having an amino acid sequence selected from the formula set forth in SEQ ID NOS:4 and 5;

B. a τ subunit having an amino acid sequence corresponding to the formula set forth in SEQ ID NO:2;

C. a e subunit having an amino acid sequence corresponding to the formula set forth in SEQ ID NO:95;

D. a α subunit including an amino acid sequence corresponding to the formula set forth in SEQ ID NO:87;

E. a β subunit having an amino acid sequence corresponding to the formula set forth in SEQ ID NO: 107; and F. combinations of the above.

The invention also extends to the genes that correspond to and can code on expression for the subunits set forth above, and accordingly includes the following: dnaX. dnaO, dnaE and dnaN. and conserved variants and active fragments thereof.

Accordingly, the Polymerase Ill-type enzyme of the present invention comprises at least one gene encoding a sub unit thereof, which gene is selected from the group consisting of dua X. dua Q, dua E and dua N, and combinations threof. More particularly, the invention extends to the nucleic acid molecule encoding them and t subunits, and includes the dua X gene which has a nucleotide sequence as set forth in SEQ ID NO. 3, as well as conserved variants, active fragments and analogs thereof. Likewise, the nucleotide sequences encoding the α sub unit (dna e gene). The e sub unit (dnaQ gene) and the β sub unit (dna N gene) each comprise the nucleo ?????? sequences as set forth respectively, in SEQ ID NO^*S: 94; 86 and 106, as well as conserved variants, active fragments and analogs thereof.

The invention also provides methods and products for identifying, isolating and cloning DNA molecules which encode such accessory subunits encoded by the recited genes of the DNA polymerase Ill-type enzyme hereof.

Yet further, the invention extends to Polymerase Ill-type enzymes prepared by the purification of an extract taken from e.g. the particular thermophile under examination, treated with appropriate solvents and then subjected to chromatographic separation on e.g. an anion exchange column, followed by analysis of long chain synthetic ability or Western analysis of the respective peaks against antibody to at least one of the anticipated enzyme subunits to confirm presence of Pol III, and thereafter, peptide sequencing of subunits that co purify and amplification to obtain the putative gene and itss encoded enzyme.

The present invention also relates to recombinant γ. τ, e, a and β subunits from thermophiles. In the instance of the γ and τ subunits, the invention includes the characterization of a frameshifting sequence that is internal to the gene and specifies relative abundance of the γ and τ gene products of dnaX. From this characterization it is obvious how to increase expression of either one of the subunits at the expense of the other (i.e. mutant frameshift could make all τ, simple recloning at the end of the frameshift could make exclusively γ and no τ).

In a further aspect of the present invention, DNA probes can be constructed from the DNA sequences coding for, eg. the T.th. dnaX, dnaO, dnaE, dnaA and dnaN genes, conserved variants and active fragments thereof, all as defined herein, and may be used to identify and isolate the corresponding genes coding for the subunits of DNA polymerase III holoenzyme from other thermophiles, such as those listed earlier herein. Accordingly, all chromosomal replicases (DNA Polymerase Ill-type) from thermophilic sources are contemplated and included herein.

The invention also extends to methods for identifying Polymerase Ill-type enzymes by use of the techniques of long-chain extension and elucidation of subuits with antibodies, as described herein and with reference to the examples.

The invention further extends to the isolated and purified DNA Polymerase III. the amino acid sequences of the γ, τ, e, and β subunits, as set forth in SEQ ID NOS:4, 5, 2, 95, 87, and 107. and the nucleotide sequences of the corresponding genes from T.th. set forth, e.g. in SEQ ID NOS:3 (dnaX), 94 (dnaO), 86 (dnaE) and 106 (dnaN), as well as to active fragments thereof, oligonucleotides and probes prepared or derived therefrom and the transformed cells that may be likewise prepared. Accordingly, the invention comprises the individual subunits enumerated above and hereinafter, corresponding isolated polynucleotides and respective amino acid sequences for each of the γ, τ, e, and β subunits, and to conserved variants, fragments, and the like, as well as to methods of their preparation and use in DNA amplification and sequencing. In a particular embodiment, the invention extends to vectors for the expression of the sub-unit genes of the present invention, and more specifically to the vectors pETlβdnaX and pET2AdnαN.

The invention also includes methods for the preparation of the DNA Polymerase III- type enzymes and the corresponding subunit genes of the present invention, and to the use of the enzymes and constructs having active fragments thereof, in the preparation, reconstitution of modification of like enzymes, as well as in amplification and sequencing of DNA by methods such as PCR. and like protocols, and to the DNA molecules amplified and sequenced by such methods. In this regard, a Pol Ill-type enzyme that is reconstituted in the absence of e, or using a mutated e with less 3'-5' exonuclease activity, may be a superior enzyme in either PCR or DNA sequencing applications, (e.g. Tabor and Richardson. 1995.) The invention is directed to methods for amplifying and sequencing a DNA molecule, particularly via the polymerase chain reaction (PCR). using the present DNA polymerase Ill-type enzymes or complexes. In particular, the invention extends to methods of amplify ing and sequencing of DNA using thermostable pol Ill-type enzyme complexes isolated from thermophilic bacteria such as Thermotoga and

Thermus species, or recombinant thermostable enzymes. The invention also provides amplified DNA molecules made by the methods of the invention, and kits for amplifying or sequencing a DNA molecule by the methods of the invention.

In this connection, the invention extends to methods for amplification of DNA that can achieve long chain extension of primed DNA, as by the application and use of Polymerase Ill-type enzymes of the present invention. An illustration of such methods is presented in Examples 13 and 14, infra.

Likewise, kits for amplification and sequencing of such DNA molecules are included, which kits contain the enzymes of the present invention, including subunits thereof, together with other necessary or desirable reagents and materials, and directions for use. The details of the practice of the invention as set forth above and later on herein, and with reference to the patents and literature cited herein, are all expressly incorporated herein by reference and made a part hereof.

As stated, and in accordance with a principal object of the present invention, Polymerase Ill-type enzymes and their sub-units are provided that are derived from thermophiles and that are adapted to participate in improved DNA amplification and sequencing techniques, and the consequent ability to prepare larger DNA strands more rapidly and accurately.

It is a further object of the present invention to provide DNA molecules that are amplified and sequenced using the Polymerase Ill-type enzymes hereof. It is a still further object of the present invention to provide enzymes and corresponding methods for amplification and sequencing of DNA that can be practiced without the participation of the clamp-loading component of the enzyme.

It is a still further object of the present invention to provide kits and other assemblies of materials for the practice of the methods of amplification and sequencing as aforesaid, that include and use the DNA polymerase Ill-type enzymes herein as part thereof.

Other objects and advantages will become apparent from a review of the ensuing description which proceeds with reference to the following illustrative drawings.

DESCRIPTION OF THE DRAWINGS

FIGURE 1 is a schematic depiction of the structure and components of enzymes of the general family to which the enzymes of the present invention belong.

FIGURE 2. Alignment of the N-terminal regions of E. coli and B. subtilis dnaXgem product - Asterisks indicate identities. The ATP binding consensus sequence is indicated. The two regions used for PCR primer design are shown in bold.

FIGURE 3. Southern analysis of T. thermophilus genomic DNA - Genomic DNA was analyzed for presence of the DnaZ gene using the PCR radiolabelled probe. Enzymes used for digestion are shown above each lane. The numbering to the right corresponds to the length of DNA fragments (kb).

FIGURES 4A and 4B depict the full sequence of the dnaX gene of T. thermophilus - DNA sequence (upper case, and corresponding to SEQ ID NO: l) and predicted amino acid sequence (lower case, and corresponding to SEQ ID NO:2) yields a 529 amino acid protein (τ) of 58.0 kDa. A putative frameshifting sequence containing several A residues 1478-1486 (underlined) may produce a smaller protein (γ) of 49.8 kDa. The potential Shine-Dalgarno (S.D.) signal is bold and underlined. The start codon is in bold, and the stop codon for τ is marked by an asterisk. The potential stop codon for γ is shown in bold after the frameshift site, and two potential Shine-Dalgarno sequences upstream of the frameshift site are indicated. Sequences of the primers used for PCR are shown in italics above the nucleotide sequence of dnaX. The ATP binding site is indicated, and the asterisks above the four Cys residues near the ATP site indicate the putative Zn++ finger. The proline rich area is indicated above the sequence. Numbering of the nucleotide sequence is presented to the right. Numbering of the amino acid sequence of τ is shown in parenthesis to the right.

FIGURE 4C depicts the isolated DNA coding sequence for the dnaX gene (also present in FIGURES 3A and 3B) in accordance with the invention, and corresponds to SEQ ID NO:3.

FIGURE 4D depicts the polypeptide sequence of the γ subunit of the Polymerase III of the present invention, and corresponds to SEQ ID NO:4.

FIGURE 4E depicts the polypeptide sequence of the γ subunit of the Polymerase III of the present invention defined by a -1 frameshift. and corresponds to SEQ ID NO:4.

FIGURE 4F depicts the polypeptide sequence of the γ subunit of the Polymerase III of the present invention defined by a -2 frameshift. and corresponds to SEQ ID NO:5.

FIGURE 5. Alignment of the γ/τ ATP binding domains for different bacteria - Dots indicate those residues that are identical to the E. coli dnaX sequence. The ATP consensus site is underlined, and the conserved cysteine residues that form the zinc finger are indicated with asterisks. E. coli. Escherichia coli; H. inf., Haemophilus influenzae; B. sub., Bacillus subtilis; C. cres., Caulobacter crescentus; M. gen.. Mycoplasma genitalium; T.th.. Thermus thermophilus. Alignments were produced using Clustel.

FIGURE 6. Signal for ribosomal frameshifting in T.th. dnaX - The diagram shows part of the sequence of the RNA around the frameshifting site, including the suspected slippery sequence A9 (bold italic). The stop codon in the -2 reading frame is indicated. Also indicated are potential step loop structures and the nearest stop codons in the -1 reading frame.

FIGURE 7. Analysis of γ and τ in T.th. cells by Western - Whole cells were lysed in SDS and electrophoresed on a 10 % SDS polyacrylamide gel then transferred to a membrane and probed with polyclonal antibody against E. coli γ/τ as described in Experimental Procedures. Positions of molecular weight size markers are shown to the left. Putative T.th. γ and τ are indicated to the right.

FIGURE 8. The frameshift sequence in T.th. dnaX promotes -1 and -2 frameshifts in E. coli - The region of the dnaX gene slippery sequence was cloned into the lacZ gene of pUC19 in three reading frames, then transformed into E. coli cells and plated on LB plates containing X-gal. The slippery sequence was also mutated by inserting two G residues into the A9 sequence and then cloned into pUC19 in all three reading frames. Color of colonies observed are indicated by the plus signs. The picture shows the colonies, the type of frameshift required for readthrough (blue color) is indicted next to the sector.

FIGURE 9. Construction of the T.th. γ/τ expression vector - A genomic fragment containing a partial sequence of dnaX was cloned into pALTER-1. This fragment was subcloned into pUC19 (pUC 19 _dnαX). Then the N-terminal section of dnαX was amplified such that the fragment was flanked by Ndel (at the initiating codon) and the internal BamHI site. This fragment was inserted to form the entire coding sequence of the dnαX gene in pUC 19 (pUC 19dnαX). The dnαX gene was then cloned behind the polyhistidine leader in the T7 based expression vector pET16 to give pET\6dnaX. Details are in "Experimental Procedures".

FIGURE 10. Purification of recombinant T.th. γ and τ subunits - T.th. γ and τ subunits were expressed in E. coli harboring pETlβdnaX. Molecular size markers are shown to the left of the gels, and the two induced proteins are labelled as g and t to the right of the gel. Panel A) 10% SDS gel of E. coli whole cell lysates before and after induction with IPTG. Panel B) 8% SDS gel of the purification two steps after cell lysis. First lane: the lysate was applied to a HiTrap Nickel chromatography column. Second lane: the T.th. γ/τ subunits were further purified on a Superose 12 gel filtration column. Third lane, the E. coli γ and τ subunits. Panel C) Western analysis of the pure T.th. γ and τ subunits (first lane) and E. coli γ and τ subunits (second lane).

FIGURE 1 1. Gel filtration of T.th. γ and τ - T.th. γ and τ were gel filtered on a Superose 12 column. Column fractions were analyzed for ATPase activity and in a Coomassie Blue stained 10% SDS polyacrylamide gel. Positions of molecular weight markers are shown to the left of the gel. The elution position of size standards analyzed in a parallel Superose 12 column under identical conditions are indicated above the gel. Thyroglobin (670 kDa), bovine gamma globin (150 kDa), chicken ovalbumin (44 kDa). equine myoglobin (17 kDa).

FIGURE 12. Characterization of the T.th. γ and τ ATPase activity - The T.th. γ/τ and E. coli τ subunits are compared in their ATPase activity characteristics. Due to the greater activity of E. coli τ. the values are plotted as percent for ease of comparison. Actual specific activities for 100 % values are given below as pmol ATP hydrolyzed/30 min./pmol T.th. γ/τ (or pmol E. coli τ). Panel A) T.th. γ and τ ATPase is stimulated by the presence of ssDNA. T.th. γ/τ was incubated at 65° C. Specific activity was: 1 1.5 (+DNA); 2.5 (-DNA): E. coli τ was assayed at 37°C. Specific activity values were: 1 12.5 (+DNA); (7.3-DNA). Panel B) Temperature stability of DNA stimulated ATPase activity. T.th. γ/τ, 1 1.3 (65°C); E. coli τ, 97.5 (37°C). Panel C) Stability of T.th. γ/τ ATPase to NaCl. T.th. γ/τ. 8.1 (100 mM added NaCl and 65oC : E. coli τ. 52.7 (0 M added NaCl and 37 °C).

FIGURES 13A-13C are graphs that summarize the purification of the DNA polymerase III from T.th. extracts. A) shows the activity and total protein in column fractions from the Heparin Agarose column. Peak 1 fractions were chromatographed on ATP agarose. and Panel B) depicts the ATP-agarose column step, and Panel C) shows the total protein and DNA polymerase activity eluted from the MonoQ column.

FIGURE 14 is a 12% SDS polyacrylamide gel stained with Coomassie Blue (Panel A) of the MonoQ column. Loud stands for the material loaded onto the column (ATP agarose bound fractions). FT stands for protein that flowed through the MonoQ column. Fractions are indicated above the gel. T.th. subunits α, τ, γ. δ, δ' in fractions 17-19 are indicated by the labels placed between fractions 18 and 19. Additional small subunits may be present but difficult to visualize, or may have mn off the gel. E. Coli, γ. δ shows a mixture of the α. γ and δ subunits of DNA polymerase III holoenzyme (they are labelled to the right in the figure). Panel B shows the Western results of an SDS gel of the MonoQ fractions probed with rabbit antiserum raised against the E. coli a subunit. L and FT are as described in Panel A. Fraction numbers are shown above the gel. The band that comigrates with E. coli alpha, and the band in the Coomassie Blue stained gel in Panel A, is marked with an arrow. This band was analyzed for microsequence and the results are shown in Fig. 15.

FIGURE 15 shows the alignments of the peptides obtained from T.th. a subunit, TTH1 (shown in A) and TTH2 (shown in B) with the amino acid sequences of the α subunits of other organisms. The amino acid number of these regions within each respective protein sequence are shown to the right. The abbreviations of the organisms are as follows. E.coli - Escheήchia coli. V.chol- Vibrio cholerae. H.inf. - Haemophilus influenzae, R.prow. - Rickettsia prowazekii, H.pyl. - Helicobacter pylori. S.sp. - Synechocystis sp., M.tub. - Mycobacterium tuberculosis, T.th. - Thermus thermophilus.

FIGURE 16 shows a partial nucleotide (Panel A) and amino acid (Panel B) sequence of the dnaE gene encoding the subunit of DNA polymerase III holoenzyme. The peptide sequence in bold was obtained by microsequencing of the subunit isolated from T.th. cells.

FIGURE 17 shows an alignment of the amino acid sequence of e subunits encoded by dnaQ of several organisms. The amino acid sequence of the Thermus thermophilus e subunit oϊ dnaQ is also shown. T.th, Thermus thermophilus; D.rad., Deinococcus radiodurans; Bac.sub., Bacillus subtilis; H.inf, Haemophilus influenzae; E.c,

Escherichia coli; H.pyl. , Helicobacter pylori. The regions used to obtain the inner part of the dnaQ gene are shown in bold. The starts used for expression of the T.th. e subunit are marked.

FIGURE 18 shows the nucleotide (Panel A) and amino acid (Panel B) sequence of the dnaQ gene encoding the e subunit of DNA polymerase III holoenzyme.

FIGURE 19 shows an alignment of the DnaA protein of several organisms. The amino acid sequence of the Thermus thermophilus DnaA protein of is also shown. T.th., Thermus thermophilus; Bac.sub., Bacillus subtilis; E.c, Escherichia coli; H.pyl, Helicobacter pylori; M. tub; Mycobacterium tuberculosis; T. mar., Thermatoga maritima.

FIGURE 20 shows the nucleotide (Panel A) and amino acid (Panel B) sequence of the dnaA gene of Thermus thermophilus.

FIGURE 21 shows the nucleotide (Panel A) and amino acid (Panel B) sequence of the dnaN gene encoding the β subunit of DNA polymerase III holoenzyme. FIGURE 22 shows an alignment of the β subunit of T.th. to the β subunits of other organisms. T.th.. Thermus thermophilus: E. coli. Escherichia coli; P. put., Pseudomonas putida P. mirab. Proteus mirabilis: H. in/1. Haemophilus influenzae; B. cap. , Buchnera aphidicola.

FIGURE 23 is a map of the pET24:dnaN plasmid. The functional regions of the plasmid are indicated by arrows and italic, restriction sites are marked with bars and symbols. The hatched parts in the plasmid correspond to T.th. dnaN.

FIGURE 24 shows the induction of T.th. β in E. coli cells harboring the T.th. β expression vector. Panel A is the cell induction. The first lane shows melecular weight markers (MW). The second lane shows uninduced E. coli cells, and the third lane shows induced E. coli. The induced T.th. β is indicated by the arrow shown to the left. Induced cells were lysed then treated with heat and the soluble portion was chromatographed on MonoQ. Panel B shows the results of MonoQ purification of T.th. β.

FIGURE 25 A is a schematic depiction of the use of the use of the enzymes of the present invention in accordance with an alternate embodiment hereof. In this scheme the clamp (β or PCNA) slides over the end of linear DNA to enhance the polymerase (Pol Ill-type such as Pol III. Polβ or Polδ.) In this fashion the clamp loader activity is not needed.

FIGURE 25B graphically demonstrates the results of the practice of the alternate embodiment of the invention described and set forth in Example 13. infra.. Lane 1, E. coli Pol III without β; Lane 2, E. coli with β; Lane 3. human Polδ without PCNA;

Lane 4, human Polδ with PCNA; Lane 5. T.th. Pol III heparin Peak 1 without T.th. β;

Lane 6, T.th. Pol III with T.th. ^β. The respective pmol synthesis in lanes 1-6 are: 6, 35, 2, 24, 0.6 and 1.9. FIGURE 26 shows the use of T.th. Pol III in extending singly primed M13mpl8 to an RFII form. The scheme at the top shows the primed template in which a DNA 57mer was a nealled to the M13mpl 8 ssDNA circle. Then T.th. β subunit (produced recombinantly) and T.th. Pol III were added to the DNA in the presence of radioactive nucleoside triphosphates. In panel B. the products of the reaction were analyzed in a 0.8% native agarose gel. The position of ssDNA starting material, the RFII product, and of intermediate species, are shown to the sides of the gel. Lane 1 , use of Pol III from the Heparin Agarose peak 1. Lane 2. use of the non-Pol III DNA polymerase contained in the peak 2 of the T.th. Heparin Agarose column.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al. "Molecular Cloning: A Laboratory Manual" (1989); "Current Protocols in Molecular Biology" Volumes I-III [Ausubel, R. M.. ed. (1994)]; "Cell Biology: A Laboratory Handbook" Volumes I-III [J. E. Celis, ed. (1994)]; "Current Protocols in Immunology" Volumes I-III [Coligan. J. E., ed. (1994)]; "Oligonucleotide Synthesis" (M.J. Gait ed. 1984): "Nucleic Acid Hybridization" [B.D. Hames & S.J. Higgins eds. (1985)]; "Transcription And Translation" [B.D. Hames & S.J. Higgins, eds. (1984)]; "Animal Cell Culture" [R.I. Freshney, ed. (1986)]; "Immobilized Cells And Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning" (1984).

Therefore, if appearing herein, the following terms shall have the definitions set out below.

The terms "DNA Polymerase III," "Polymerase Ill-type enzyme(s)", "Polymerase III enzyme complex(s)". "T.th. DNA Polymerase III", "clamp loader" and any variants not specifically listed, may be used herein interchangeably, as are β subunit and sliding clamp and clamp as are also γ complex, clamp loader and RFC. as used throughout the present application and claims refer to proteinaceous material including single or multiple proteins, and extends to those proteins having the amino acid sequence data described herein and presented in the Figures and corresponding Sequence Listing entries, and the corresponding profile of activities set forth herein and in the Claims. Accordingly, proteins displaying substantially equivalent or altered activity are likewise contemplated. These modifications may be deliberate, for example, such as modifications obtained through site-directed mutagenesis, or may be accidental, such as those obtained through mutations in hosts that are producers of the complex or its named subunits. Also, the terms "DNA Polymerase III," " T.th. DNA Polymerase III," and "γ and τ subunits", "β subunit", "α subunit", "e subunit", "sliding clamp" and "clamp loader" are intended to include within their scope proteins specifically recited herein as well as all substantially homologous analogs and allelic variations.

Also as used herein, the term "thermolabile enzyme" refers .to a DNA polymerase which is not resistant to inactivation by heat. For example, T5 DNA polymerase. the activity of which is totally inactivated by exposing the enzyme to a temperature of 90 °C for 30 seconds, is considered to be a thermolabile DNA polymerase. As used herein, a thermolabile DNA polymerase is less resistant to heat inactivation than in a thermostable DNA polymerase. A thermolabile DNA polymerase typically will also have a lower optimum temperature than a thermostable DNA polymerase. Thermolabile DNA polymerases are typically isolated from mesophilic organisms, for example mesophilic bacteria or eukaryotes, including certain animals.

As used herein, the term "thermostable enzyme" refers to an enzyme which is stable to heat and is heat resistant and catalyzes (facilitates) combination of the nucleotides in the proper manner to form the primer extension products that are complementary to each nucleic acid strand. Generally, the synthesis will be initiated at the 3' end of each primer and will proceed in the 5' direction along the template strand, until synthesis terminates, producing molecules of different lengths.

The thermostable enzyme herein must satisfy a single criterion to be effective for the amplification reaction, i.e., the enzyme must not become irreversibly denatured (inactivated) when subjected to the elevated temperatures for the time necessary to effect denaturation of double-stranded nucleic acids. Irreversible denaturation for purposes herein refers to permanent and complete loss of enzymatic activity. The heating conditions necessary for denaturation will depend, e.g., on the buffer salt concentration and the length and nucleotide composition of the nucleic acids being denatured, but typically range from about 90° to about 96 °C for a time depending mainly on the temperature and the nucleic acid length, typically about 0.5 to four minutes. Higher temperatures may be tolerated as the buffer salt concentration and/or GC composition of the nucleic acid is increased. Preferably, the enzyme will not become irreversibly denatured at about 90°-100°C.

The thermostable enzymes herein preferably have an optimum temperature at which they function that is higher than about 40 °C, which is the temperature below which hybridization of primer to template is promoted, although, depending on (1) magnesium and salt concentrations and (2) composition and length of primer, hybridization can occur at higher temperature (e.g.. 45°-70°C). The higher the temperature optimum for the enzyme, the greater the specificity and/or selectivity of the primer-directed extension process. However, enzymes that are active below 40°C, e.g., at 37 °C, are also within the scope of this invention provided they are heat-stable. Preferably, the optimum temperature ranges from about 50° to 90°C. more preferably 60°-80°C. In this connection, the term "elevated temperature" as used herein is intended to cover sustained temperatures of operation of the enzyme that are equal to or higher than about 60 °C. The term "template" as used herein refers to a double-stranded or single-stranded DNA molecule which is to be amplified, synthesized or sequenced. In the case of a double-stranded DNA molecule, denaturation of its strands to form a first and a second strand is performed before these molecules may be amplified, synthesized or sequenced. A primer, complementary to a portion of a DNA template is hybridized under appropriate conditions and the DNA polymerase of the invention may then synthesize a DNA molecule complementary to said template or a portion thereof. The newly synthesized DNA molecule, according to the invention, may be equal or shorter in length than the original DNA template. Mismatch incorporation during the synthesis or extension of the newly synthesized DNA molecule may result in one or a number of mismatched base pairs. Thus, the synthesized DNA molecule need not be exactly complementary to the DNA template.

The term "incoφorating" as used herein means becoming a part of a DNA molecule or primer.

As used herein "amplification" refers to any in vitro method for increasing the number of copies of a nucleotide sequence, or its complimentary sequence, with the use of a DNA polymerase. Nucleic acid amplification results in the incorporation of nucleotides into a DNA molecule or primer thereby forming a new DNA molecule complementary to a DNA template. The formed DNA molecule and its template can be used as templates to synthesize additional DNA molecules. As used herein, one amplification reaction may consist of many rounds of DNA replication. DNA amplification reactions include, for example, polymerase chain reactions (PCR). One PCR reaction may consist of 30 to 100 "cycles" of denaturation and synthesis of a DNA molecule. In this connection, the use of the term "long stretches of DNA" as it refers to the extension of primer along DNA is intended to cover such extrusions of an average length exceeding 7 kilobases. Naturally, such length will vary, and all such variations are considered to be included within the scope of the invention. As used herein, the term "holoenzyme" refers to a multi-subunit DNA polymerase activity comprising and resulting from various subunits which each may have distinct activities but which when contained in an enzyme reaction operate to carry out the function of the polymerase (typically DNA synthesis) and enhance its activity over use of the DNA polymerase subunit alone. For example, E. coli DNA polymerase III is a holoenzyme comprising three components of one or more subunits each: (1) a core component consisting of a heterotrimer of . ε and θ subunits; (2) a β component consisting of a β subunit dimer; and (3) a γ clex component consisting of a heteropentamer of γ. δ. δ', χ and Ψ subunits (see Studwell, P.S., and O'Donnell, M., J. Biol. Chem. 265(2): 1 171-1 178 (1990), for review). These three components, and the various subunits of which they consist, are linked non-covalently to form the DNA polymerase III holoenzyme complex.

As used herein, "enzyme complex" refers to a protein structure consisting essentially of two or more subunits of a holoenzyme. which may or may not be identical, noncovalently linked to each other to form a multi-subunit .structure. An enzyme complex according to this definition ideally will have a particular enzymatic activity, up to and including the activity of the holoenzyme. For example, a "DNA pol III enzyme complex" as used herein means a multi-subunit protein activity comprising two or more of the subunits of the DNA pol III holoenzyme as defined above, and having DNA polymerizing or synthesizing activity. Thus, this term encompasses the native holoenzyme. as well as an enzyme complex lacking one or more of the subunits of the holoenzyme (e.g., DNA pol III exo-, which lacks the ε subunit).

The amino acid residues described herein are preferred to be in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired fuctional property of immunoglobulin-binding is retained by the polypeptide. NH₂ refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. In keeping with standard polypeptide J nomenclature, J. Biol. Chem., 243:3552-59 (1969). abbreviations for amino acid residues are shown in the following Table of Correspondence:

TABLE OF CORRESPONDENCE

SYMBOL AMINO ACID

1 -Letter 3 -Letter

Y Tyr tyrosine

G Gly glycine

F Phe phenylalanine

M Met methionine

A Ala alanine

S Ser serine

I Ile isoleucine

L Leu leucine

T Thr threonine

V Val . valine

P Pro proline

K Lys lysine

H His histidine

Q Gin glutamine

E Glu glutamic acid

W Trp tryptophan

R Arg arginine

D Asp aspartic acid

N Asn asparagine

C Cys cysteine

It should be noted that all amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino- terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues. The above Table is presented to correlate the three-letter and one-letter notations which may appear alternately herein.

A "replicon" is any genetic element (e.g.. plasmid. chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.

A "vector" is a replicon, such as plasmid. phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.

A "DNA molecule" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine. or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double- stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids. and chromosomes. In discussing the structure of particular double- stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).

An "origin of replication" refers to those DNA sequences that participate in DNA synthesis.

A DNA "coding sequence" is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences. cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA. and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.

Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.

A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease S I), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Prokaryotic promoters contain Shine- Dalgarno sequences in addition to the -10 and -35 consensus sequences.

An "expression control sequence" is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.

A "signal sequence" can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.

The term "oligonucleotide," as used generally herein, such as in referring to probes prepared and used in the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.

The term "primer" as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e.. in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.

The primers herein are selected to be "substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.

As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.

A cell has been "transformed" by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In prokaryotes. yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited-by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.

Two DNA sequences are "substantially homologous" when at least about 75%

(preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.

It should be appreciated that also within the scope of the present invention are DNA sequences encoding T.th. DNA Polymerase III which code for a T.th. DNA Polymerase III having the same amino acid sequence as SEQ ID NO:2, but which are degenerate to SEQ ID NO:2. By "degenerate to" is meant that a different three-letter codon is used to specify a particular amino acid. It is well known in the art that the following codons can be used interchangeably to code for each specific amino acid:

Phenylalanine (Phe or F) UUU or UUC Leucine (Leu or L) UUA or UUG or CUU or CUC or CUA or CUG

Isoleucine (Ile or I) AUU or AUC or AUA

Methionine (Met or M) AUG

Valine (Val or V) GUU or GUC of GU A or GUG

Serine (Ser or S) UCU or UCC or UCA or UCG or AGU or AGC Proline (Pro or P) CCU or CCC or CCA or CCG

Threonine (Thr or T) ACU or ACC or ACA or ACG

Alanine (Ala or A) GCU or GCG or GCA or GCG

Tyrosine (Tyr or Y) UAU or UAC

Histidine (His or H) CAU or CAC Glutamine (Gin or Q) CAA or CAG

Asparagine (Asn or N) AAU or AAC

Lysine (Lys or K) AAA or AAG

Aspartic Acid (Asp or D) GAU or GAC

Glutamic Acid (Glu or E) GAA or GAG Cysteine (Cys or C) UGU or UGC

Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG

Glycine (Gly or G) GGU or GGC or GGA or GGG

Tryptophan (Trp or W) UGG Termination codon UAA (ochre) or UAG (amber) or UGA (opal)

It should be understood that the codons specified above are for RNA sequences. The corresponding codons for DNA have a T substituted for U.

Mutations can be made, e.g. in SEQ ID NO: 1, or any of the nucleic acids set forth herein, such that a particular codon is changed to a codon which codes for a different amino acid. Such a mutation is generally made by making the fewest nucleotide changes possible. A substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non-conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping). Such a conservative change generally leads to less change in the structure and function of the resulting protein. A non-conservative change is more likely to alter the structure, activity or function of the resulting protein. The present invention should be considered to include sequences containing conservative changes which do not significantly alter the activity or binding characteristics of the resulting protein.

The following is one example of various groupings of amino acids:

Amino acids with nonpolar R groups

Alanine Valine Leucine Isoleucine Proline Phenylalanine

Tryptophan

Methionine

Amino acids with uncharged polar R groups

Glycine

Serine

Threonine

Cysteine

Tyrosine Asparagine

Glutamine

Amino acids with charged polar R groups (negatively charged at ph 6.0)

Aspartic acid Glutamic acid

Basic amino acids (positively charged at pH 6.0)

Lysine

Arginine

Histidine (at pH 6.0)

Another grouping may be those amino acids with phenyl groups:

Phenylalanine Tryptophan Tyrosine Another grouping may be according to molecular weight (i.e., size of R groups):

Glycine 75

Alanine 89

Serine 105

Proline 115

Valine 117

Threonine 119

Cysteine 121

Leucine 131

Isoleucine 131

Asparagine 132

Aspartic acid 133

Glutamine 146

Lysine 146

Glutamic acid 147

Methionine 149

Histidine (at pH 6.0) 155

Phenylalanine 165

Arginine 174

Tyrosine 181

Tryptophan 204

Particularly preferred substitutions are:

- Lys for Arg and vice versa such that a positive charge may be maintained;

- Glu for Asp and vice versa such that a negative charge may be maintained; - Ser for Thr such that a free -OH can be maintained; and

- Gin for Asn such that a free NH, can be maintained. Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property. For example, a Cys may be introduced into a potential site for disulfide bridges with another Cys. A His may be introduced as a particularly "catalytic" site (i.e.. His can act as an acid or base and is the most common amino acid in biochemical catalysis). Pro may be introduced because of its particularly planar structure, which induces β-turns in the protein's structure.

Two amino acid sequences are "substantially homologous" when at least about 70% of the amino acid residues (preferably at least about 80%, and most preferably at least about 90 or 95%) are identical, or represent conservative substitutions.

A "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. Another example of a-heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally- occurring mutational events do not give rise to a heterologous region of DNA as defined herein.

An "antibody" is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope. The term encompasses polyclonal, monoclonal, and chimeric antibodies, the last mentioned described in further detail in U.S. Patent Nos. 4,816,397 and 4,816,567.

An "antibody combining site" is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen. J

The phrase "antibody molecule" in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule.

Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope, including those portions known in the art as Fab, Fab', F(ab')₂ and F(v). which portions are preferred for use in the therapeutic methods described herein.

Fab and F(ab')₂ portions of antibody molecules are prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibody molecules by methods that are well-known. See for example. U.S. Patent No. 4.342,566 to Theofilopolous et al. Fab' antibody molecule portions are also well-known and are produced from F(ab')₂ portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoefhanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide. An antibody containing intact antibody molecules is preferred herein.

The phrase "monoclonal antibody" in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular antigen. A monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts. A monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen; e.g., a bispecific (chimeric) monoclonal antibody.

A DNA sequence is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence. The term "operatively linked" includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.

The term "standard hybridization conditions" refers to salt and temperature conditions substantially equivalent to 5 x SSC and 65 °C for both hybridization and wash. However, one skilled in the art will appreciate that such "standard hybridization conditions" are dependent on particular conditions including the concentration of sodium and magnesium in the buffer, nucleotide sequence length and concentration, percent mismatch, percent formamide. and the like. Also important in the determination of "standard hybridization conditions" is whether the two sequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standard hybridization conditions are easily determined by one skilled in the art according to well known formulae, wherein hybridization is typically 10-20°C below the predicted or determined T_m with washes of higher stringency, if desired.

In its primary aspect, the present invention concerns the identification of a class of DNA Polymerase Ill-type enzymes or complexes found in thermophilic bacteria such as Thermus thermophilus (T.th.), and other eubacteria such as Thermatoga, which exhibit the following characteristics, among their properties: the ability to extend a primer over a long stretch of ssDNA at elevated temperature, stimulation by its cognate sliding clamp of the type that is assembled on DNA by a clamp loader (e.g. γ complex), accessory subunits that exhibit DNA-stimulated ATPase activity at elevated temperature and/or ionic strength, and an associated 3'-5' exonuclease activity. In a particular aspect, the invention extends to Polymerase Ill-type enzymes derived from a broad class of thermophilic bacteria that include polymerases isolated from the thermophilic bacteria Thermus thermophilus (T.th. polymerase). Thermococcus litoralis (Tli or VENT™ polymerase). Pyrococcus furiosus (Pfu or DEEPVENT polymerase). Pyrococcus woosii (Pwo polymerase) and other Pyrococcus species, Bacillus ster other mophilus (Bst polymerase), sulfolobus acidocaldarius (Sac polymerase). thermoplasma acidophilum (Tac polymerase), Thermus favus (Tfl/Tub polymerase), Thermus ruber (Tru polymerase), Thermus brockianus (DYNAZYME™ polymerase). Thermotoga neapolitana (Tne polymerase; See WO 96/10640). Thermotoga maritima (Tma polymerase; See U.S. Patent No. 5,374,553) and other species of the Thermotoga genus (Tsp polymerase) and Methanobacterium thermoautotrophicum (Mth polymerase). The particular polymerase discussed herein by way of illustration and not limitation, is the enzyme derived from T. th..

Polymerase Ill-type enzymes covered by the invention include those that may be prepared by purification from cellular material, as described in detail in Example 9 herein, as well as enzyme assemblies or complexes that comprise the combination of individually prepared enzyme subunits or components. Accordingly, the entire enzyme may be prepared by purification from cellular material, or may be constructed by the preparation of the individual components and their assembly into the functional enzyme. A representative and non-limitative protocol for the preparation of an enzyme by this latter route is set forth in U.S. Patent No. 5,583,026, issued December 10, 1996, to one of the inventors herein, and the disclosure thereof is incoφorated herein in its entirety for such purpose.

Likewise, individual subunits may be modified, e.g. as by incoφoration therein of single residue substitutions to create active sites therein, for the puφose of imparting new or enhanced properties to enzymes containing the modified subunits. See, for example, Tabor, S. et al. (1995) Proc. Natl. Acad. Sci. USA, 92(14):6339-6343, the disclosure of which is also incoφorated herein in its entirety. Likewise, individual subunits prepared in accordance with the invention, may be used individually and for example, may be substituted for their counteφarts in other enzymes, to improve or particularize the properties of the resultant modified enzyme. Such modifications are within the skill of the art and are considered to be included within the scope of the present invention.

Accordingly, the invention includes the various subunits that may comprise the enzymes, and accordingly extends to the genes and corresponding proteins that may be encoded thereby, such as the , β, γ, e, τ, δ and δ' subunits, respectively. More particularly, the subunit corresponds to dnaE, the β subunit corresponds to dnaN, the e subunit corresponds to dnaQ, and the γ and τ subunits correspond to dnaX.

Accordingly, the Polymerase Ill-type enzyme of the present invention comprises at least one gene encoding a sub unit thereof, which gene is selected from the group consisting of dua X. dua Q. dua E and dua N. and combinations threof. More particularly, the invention extends to the nucleic acid molecule encoding them and t subunits, and includes the dua X gene which has a nucleotide sequence as set forth in SEQ ID NO. 3, as well as conserved variants, active fragments and analogs thereof. Likewise, the nucleotide sequences encoding the a sub unit (dna e gene). The e sub unit (dnaQ gene) and the β sub unit (dna N gene) each comprise the nucleo ?????? sequences as set forth respectively, in SEQ ID NO^'S: 94; 86 and 106. as well as conserved variants, active fragments and analogs thereof.

A particular Polymerase Ill-type enzyme in accordance with the invention may include at least one of the following sub-units:

A. a γ subunit having an amino acid sequence selected from the formula set forth in SEQ ID NOS:4 and 5; B. a τ subunit having an amino acid sequence corresponding to the formula set forth in SEQ ID NO:2;

C. a e subunit having an amino acid sequence corresponding to the formula set forth in SEQ ID NO: 95; D. a subunit including an amino acid sequence corresponding to the formula set forth in SEQ ID NO:87;

The invention also includes and extends to the use and application of the enzyme and/or one or more of its components for DNA molecule amplification and sequencing by the methods set forth hereinabove, and in greater detail later on herein.

One of the subunits of the invention is the γ/τ subunit encoded by a dnaX gene, which frameshifts as much as -2 with high efficiency, and that, upon frameshifting, leads to the addition of more than one extra amino acid residue to the C-terminus (to form the γ subunit). Further, the invention likewise extends to a dnaX gene derived from a hermophile such as T.th., that possesses the frameshift defined herein and that codes for expression of the γ and τ subunits of DNA Polymerase III.

The present invention provides methods for amplifying or sequencing a nucleic acid molecule comprising contacting the nucleic acid molecule with a composition comprising a DNA polymerase III enzyme (DNA pol III) complex, preferably a DNA pol III complex that is substantially reduced in 3'-5' exonuclease activity. DNA pol III complexes used in the methods of the present invention are thermostable.

The invention also provides DNA molecules amplified by the present methods, methods of preparing a recombinant vector comprising inserting a DNA molecule amplified by the present methods into a vector, which is preferably an expression vector, and recombinant vectors prepared by these methods. The invention also provides methods of preparing a recombinant host cell comprising inserting a DNA molecule amplified by the present methods into a host cell, which preferably a bacterial cell, most preferably an Escherichia coli cell; a yeast cell; or an animal cell, most preferably an insect cell, a nematode cell or a mammalian cell. The invention also provides and recombinant host cells prepared by these methods.

In additional preferred embodiments, the present invention provides kits for amplifying or sequencing a nucleic acid molecule. DNA amplification kits according to the invention comprise a carrier means having in close confinement therein two or more container means, wherein a first container means contains a DNA polymerase III enzyme complex and a second container means contains a deoxynucleoside triphosphate. DNA sequencing kits according to the present invention comprise a multi-protein Pol Ill-type enzyme complex and a second container means contains a dideoxynucleoside triphosphate. The DNA pol III contained in the container means of such kits is preferably substantially reduced in 5'-3' exonuclease activity, may be thermostable, and may be isolated from the thermophilic cellular sources described above. Most preferably, the DNA pol III contained in the container means of such kits is a DNA polymerase Ill-type complex of a fhermophile which lacks the ε subunit.

DNA pol Ill-type enzyme complexes for use in the present invention may be isolated from any organism that produced the DNA pol Ill-type enzyme complexes naturally or recombinantly. Such enzyme complexes may be thermostable, isolated from a variety of thermophilic organisms.

The thermostable DNA polymerase Ill-type enzymes or complexes that are an important aspect of this invention, may be isolated from a variety of thermophilic bacteria that are available commercially (for example, from American Type Culture Collection. Rockville. Maryland). Suitable for use as sources of thermostable enzymes are the thermophilic bacteria Thermus aquaticus, Thermus thermophilus, Thermococcus Utoralis, Pyrococcus furiosus, Pyrococcus woosii and other species of the Pyrococcus genus. Bacillus stearothermophilus. Sulfolobus acidocaldarius. Thermoplasma acidophilum, Thermus flavus. Thermus ruber. Thermus brockianus. Thermotoga neapolitana, Thermotoga maritima and other species of the Thermotoga genus, and Methanobacterium thermoautotrophicum, and mutants of each of these species. It will be understood by one of ordinary skill in the art , however, that any thermophilic microorganism might be used as a source of thermostable DNA pol Ill- type enzymes and polypeptides for use in the methods of the present invention. Bacterial cells may be grown according to standard microbiological techniques, using culture media and incubation conditions suitable for growing active cultures of the particular thermophilic species that are well-known to one of ordinary skill in the art (see. e.g., Brock. T.D.. and Freeze, H.. J. Bacteriol. 98(l):289-297 (1969); Oshima. T.. and Imahori. K. Int. J. Syst. Bacteriol. 24(1): 102-1 12(1974)). Thermostable DNA pol III complexes may then be isolated from such thermophilic cellular sources as described for thermolabile complexes above.

As stated above and in accordance with the present invention, nucleic acid molecules may be amplified according to any of the literature-described manual or automated amplification methods. Such methods includes, but are not limited to. PCR (U.S. Patent Nos. 4,683.195 and 4.683,202). Strand Displacement Amplification (SDA; U.S. Patent No. 5,455,166; EP 0 684 315). and Nucleic Acid Sequence-Based

Amplification (NASBA; U.S. Patent No. 5,409,818; EP 0 329 822). Most preferably, nucleic acid molecules are amplified by the methods of the present invention using PCR-based amplification techniques.

In the initial steps of each of these amplification methods, the nucleic acid molecule to be amplified is contacted with a composition comprising a DNA polymerase belonging to the evolutionary "family A" class (e.g. Taq DNA pol I or E. coli pol I) or the "family "B" class (e.g. Vent and Pfii DNA polymerases ~ see Ito, J., and Braithwaite, D., Nucl. Acids Res. 19(15):4045-4057 (1991)). All of these DNA polymerases are present as single subunits and are primarily involved in DNA repair. In contrast, the DNA pol Ill-type enzymes are multisubunit complexes that mainly function in the replication of the chromosome, and the subunit containing the DNA polymerase activity is in the "family C" class.

Thus, in amplifying a nucleic acid molecule according to the methods of the present invention, the nucleic acid molecule is contacted with a composition comprising a thermostable DNA pol Ill-type enzyme complex. The DNA pol Ill-type complexes used in the present methods are preferably substantially reduced in 3'-5' exonuclease activity (i.e., they are "exo-").

Once the nucleic acid molecule to be amplified is contacted with the DNA pol Ill-type complex, the amplification reaction may proceed according to standard protocols for each of the above-described techniques. Since most of these techniques comprise a high-temperature denaturation step, if a thermolabile DNA pol Ill-type enzyme complex (such as E. coli DNA pol III exo-) is used in nucleic acid amplification by any of these techniques the enzyme would need to be added at the start of each amplification cycle, since it would be heat-inactivated at the denaturation step. However, a thermostable DNA pol Ill-type complex used in these methods need only be added once at the start of the amplification (as for Taq DNA polymerase in traditional PCR amplifications), as its activity will be unaffected by the high temperature of the denaturation step. It should be noted, however, that because DNA pol Ill-type enzymes have a much more rapid rate of nucleotide incoφoration than the polymerases commonly used in these amplification techniques, the cycle times may need to be adjusted to shorter intervals than would be standard.

In an alternative preferred embodiment, the invention provides methods of extending primers for several kilobases. a reaction that is central to amplifying large nucleic acid molecules, by a technique commonly referred to as "long PCR" (Barnes, W.M., Proc. Natl. Acad. Sci. USA 1 :2216-2220 ( 1994); Cheng. S. et al.. Proc. Natl. Acad. Sci. USA 91 :5659-5699 ( 1994)).

In such a method the target primed DNA can contain a single strand stretch of DNA to be copied into the double strand form of several or tens of kilobases. The reaction is performed in a suitable buffer, preferably Tris, at a pH of between 5.5 - 9.5, preferably 7.5. The reaction also contains MgC in the range 1 mM to 10 mM. preferably 8 mM. and may contain a suitable salt such as NaCl, KC1 or sodium or potassium acetate. The reaction also contains ATP in the range of 20 uM to 1 mM, preferably 0.5 mM, that is needed for the clamp loader to assemble the clamp onto the primed template, and a sufficient concentration of deoxynucleoside triphosphates in the range of 50μM to 0.5 mM. preferably 60 μM for chain extension. The reaction contains a sliding clamp, such as the ^β subunit, in the range of 20ng to 200 ng, preferably 100 ng, for action as a clamp to stimulate the DNA polymerase. The chain extension reaction contains a DNA polymerase and a clamp loader, that could be added either separately or as a single Pol III* -like particle, preferably as a Pol III* like particle that contains the DNA polymerase and clamp loading activities. The Pol Ill-type enzyme is added preferably at a concentrations of about 0.0002-200 units per milliliter, about 0.002-100 units per milliliter. about 0.2-50 units per milliliter, and most preferably about 2-50 units per milliliter. The reaction is incubated at elevated temperature, preferably 60 °C or more, and could include other proteins to enhance activity such as a single strand DNA binding protein.

In another preferred embodiment, the invention provides methods of extending primers on linear templates in the absence of the clamp loader. In this reaction, the primers are annealled to the linear DNA, preferably at the ends such as in standard PCR applications. The reaction is performed in a suitable buffer, preferably Tris, at a pH of between 5.5 - 9.5, preferably 7.5. The reaction also contains MgCl₂ in the range of 1 mM to 10 mM. preferably 8 mM, and may contain a suitable salt such as NaCl, KC1 or sodium or potassium acetate. The reaction also contains a sufficient concentration of deoxynucleoside triphosphates in the range of 50μM to 0.5 mM, preferably 60 μM for chain extension. The reaction contains a sliding clamp, such as the β subunit, in the range of 20ng to 20 μg, preferably 7 μg. for ability to slide on the end of the DNA and associate with the polymerase for action as a clamp to stimulate the DNA polymerase. The chain extension reaction also contains a Pol Ill-type polymerase subunit such as , core, or a Pol III* -like particle. The Pol Ill-type enzyme is added preferably at a concentrations of about 0.0002-200 units per milliliter, about 0.002-100 units per milliliter. about 0.2-50 units per milliliter. and most preferably about 2-50 units per milliliter. The reaction is incubated at elevated temperature, preferably 60 °C or more, and could include other proteins to enhance activity such as a single strand DNA binding protein.

The methods of the present invention thus will provide high-fidelity amplified copies of a nucleic acid molecule in a more rapid fashion than traditional amplification methods using the repair-type enzymes.

These amplified nucleic acid molecules may then be manipulated according to standard recombinant DNA techniques. For example, a nucleic acid molecule amplified according to the present methods may be inserted into a vector, which is preferably an expression vector, to produce a recombinant vector comprising the amplified nucleic acid molecule. This vector may then be inserted into a host cell, where it may, for example, direct the host cell to produce a recombinant polypeptide encoded by the amplified nucleic acid molecule. Methods for inserting nucleic acid molecules into vectors, and inserting these vectors into host cells, are well-known to one of ordinary skill in the art (see, e.g.. Maniatis. T.. et al., Molecular Cloning, A Laboratory Manual, Boca Raton, Florida: CRC Press (1992)).

Alternatively, the amplified nucleic acid molecules may be directly inserted into a host cell, where it may be incoφorated into the host cell genome or may exist as an extrachromosomal nucleic acid molecule, thereby producing a recombinant host cell. Methods for introduction of a nucleic acid molecule into a host cell, including calcium phosphate transfection. DEAE-dextran mediated transfection, cationic lipid-mediated transfection. electroporation. transduction. infection or other methods, are described in many standard laboratory manuals (see e.g., Davis et al., Basic Methods In Molecular Biology (1986)).

For each of the above techniques wherein an amplified nucleic acid molecule is introduced into a host cell via a vector or via direct introduction, preferred host cells include but are not limited to a bacterial cell, a yeast cell, or an animal cell. Bacterial host cells preferred in the present invention are E. coli, Bacillus spp., Streptomyces spp., Erwinia spp., Klebsiella spp. and Salmonella typhimurium. Preferred as a host cell is E. coli, and particularly preferred are E. coli strians DH10B and Stbl2, which are available commercially (Life Technologies. Inc. Gaitherburg, Maryland). Preferred animal host cells are insect cells, nematode cells and mammalian cells. Insect host cells preferred in the present invention are Drosophila spp. cells, Spodoptera Sf9 and Sf21 cells, and Trichoplusa High-Five- cells, each of which is available commercially (e.g., from Invitrogen; San Diego, California). Preferred nematode host cells are those derived from C. elegans. and preferred mammalian host cells are those derived from rodents, particularly rats, mice or hamsters, and primates, particularly monkeys and humans. Particularly preferred as mammalian host cells are CHO cells, COS cells and VERO cells.

By the present invention, nucleic acid molecules may be sequenced according to any of the literature-described manual or automated sequencing methods. Such methods include, but are not limited to, dideoxy sequencing methods ("Sanger sequencing"; Sanger, F., and Coulson, A.R.. J. Mol. Biol. 94:444-448 (1975); Sanger, F., et al, Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977); U.S. Patent Nos. 4.962,022 and 5,498,523), as well as more complex PCR-based nucleic acid fingeφrinting techniques such as Random Amplified Polymoφhic DNA 9RAPD) analysis (Williams, J.G.K. et al, Nucl. Acids Res. 18(22):6531-6535. 1990). Arbitrarily Primed PCR (AP-PCR: Welsh, J., and McClelland. M.. Nucl. Acids Res. 18(24):7213- 7218. 1990), DNA Amplification Fingerprinting (DAF; Caetano-Anolles et al.. Bio/Technology 9:553-557. 1991), microsatellite PCR or Directed Amplification of Minisatellite-region DNA (DAMD; Heath. D.D., et al., Nucl. Acids Res. 21(24):5782- 5785. 1993), and Amplification Fragment Length Polymoφhism (AFLP) analysis (EP 0 534 858; Vos. P.. et al., Nucl. Acids Res. 23(21):4407-4414, 1995; Lin. J.J., and Kuo. J.. FOCUS 17(2):66-70, 1995).

As described above for amplification methods, the nucleic acid molecule to be sequenced by these methods is typically contacted with a composition comprising a type A or type B DNA polymerase. By contrast, in sequencing a nucleic acid molecule according to the methods of the present invention, the nucleic acid molecule is contacted with a composition comprising a thermostable DNA pol Ill-type enzyme complex instead of necessarily using a DNA polymerase of the family A or B classes. As for amplification methods, the DNA pol Ill-type complexes used in the nucleic acid sequencing methods of the present invention are preferably substantially reduced in 5'-3' exonuclease activity; most preferable for use in the present methods is a DNA polymerase Ill-type complex which lacks the ε subunit. DNA pol Ill-type complexes used for nucleic acid sequencing according to the present methods are used at the same preferred concentration ranges described above for long chain extension of primers.

Once the nucleic acid molecule to be sequenced is contacted with the DNA pol III complex, the sequencing reactions may proceed according to the protocols disclosed in the above-referenced techniques.

As discussed above, the invention extends to kits for use in nucleic acid amplification or sequencing utilizing DNA polymerase Ill-type enzymes according to the present methods. A DNA amplification kit according to the present invention may comprise a carrier means, such as vials, tubes, bottles and the like. A first such container means may contain a DNA polymerase Ill-type enzyme complex, and a second such container means may contain a deoxynucleoside triphosphate. The amplification kit encompassed by this aspect of the present invention may further comprise additional reagents and compounds necessary for carrying out standard nucleic amplification protocols (See U.S. Patent Nos. 4,683,195 and 4,683,202, which are directed to methods of DNA amplification by PCR).

Similarly, a DNA sequencing kit according to the present invention comprises a carrier means having in close confinement therein two or more container means, such as vials, tubes, bottles and the like. A first such container means may contain a DNA polymerase Ill-type enzyme complex, and a second such container means may contain a dideoxynucleoside triphosphate. The sequencing kit may further comprise additional reagents and compounds necessary for carrying out standard nucleic sequencing protocols,, such as pyrophosphatase, agarose or polyacrylamide media for formulating sequencing gels, and other components necessary for detection of sequenced nucleic acids (See U.S. Patent Nos. 4,962,020 and 5,498,523, which are directed to methods of DNA sequencing).

The DNA polymerase Ill-type complex contained in the first container means of the amplification and sequencing kits provided by the invention is preferably a thermostable DNA polymerase Ill-type enzyme complex and more preferably a DNA polymerase Ill-type enzyme complex that is substantially reduced in 3-5' exonuclease activity. Naturally, the foregoing methods and kits are presented as illustrative and not restrictive of the use and application of the enzymes of the invention for DNA molecule amplification and sequencing. Likewise, the applications of specific embodiments of the enzymes, including conserved variants and active fragments thereof are considered to be disclosed and included within the scope of the invention.

As discussed earlier, individual subunits could be modified to customize enzyme construction and corresponding use and activity. For example, the region of that interacts with ^β could be subcloned onto another DNA polymerase. thereby causing β to enhance the activity of the recombinant polymerase. Alternatively, the β clamp could be modified to function with another protein or enzyme thereby enhancing its activity or acting to localize its action to a particular targeted DNA. Finally, the polymerase active site could be modified to enhance its action, sor example changing Tyrosine enabling more equal site stoppage with the four ddNTPs (Tabor et al. 1995). This represents a particular non-limiting illustration of the scope and practice of the present invention with reference to the utility of individual subunits hereof.

Accordingly and as stated above, the present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof, which encodes any one or all of the subunits of the DNA Polymerase Ill-type enzymes of the present invention, or active fragments thereof. In the instance of the τ subunit, a predicted molecular weight of about 58 kD and an amino acid sequence set forth in SEQ ID NOS:4 or 5 is comprehended; preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding the 58 kD subunit of the

Polymerase III of the invention, that has a nucleotide sequence or is complementary to a DNA sequence shown in FIGURES 4A and 4B (SEQ ID NO: 1), and the coding region for dnaXsei forth in FIGURE 4C (SEQ ID NO:3). The γ subunit is smaller, and is approximately 50 kD, depending upon the extent of the frameshift that occurs. More particularly, and as set forth in FIGURE 4E (SEQ ID NO:4), the γ subunit defined by a -1 frameshift possesses a molecular weight of 50.8 kD, while the γ subunit defined by a -2 frameshift, set forth in FIGURE 4F (SEQ ID NO:5), possesses a molecular weight of 49.8 kD.

As discussed above, the invention also extends to the genes including dnaX, dnaQ, dnaE and dnaN, that have been isolated and purified from Thermus thermophilus, to corresponding vectors for the genes, and particularly, to the vectors pETdnaX and pETdnaN. and to host cells including such vectors. In this connection, probes have been prepared which hybridize to the DNA polymerase Ill-type enzymes of the present invention, and which are selected from the group consisting of the oligonucleotide defined in SEQ ID NO:6; the oligonucleotide defined in SEQ ID NO:8; the oligonucleotide defined in SEQ ID NO: 10; the oligonucleotide defined in SEQ ID NO: l 1 ; the oligonucleotide defined in SEQ ID NO: 12; the oligonucleotide defined in SEQ ID NO: 13; the oligonucleotide defined in SEQ ID NO:14; the oligonucleotide defined in SEQ ID NO: 15, and the oligonucleotide defined in SEQ ID NO: 16.

The methods of the invention include a method for producing a recombinant thermostable DNA polymerase Ill-type enzyme from a thermophilic bacterium such as Thermus thermophilus which comprises culturing a host cell transformed with a vector of the invention under conditions suitable for the expression of the present DNA polymerase III. Another method includes a method for isolating a target DNA fragment consisting essentially of a DNA coding for a thermostable DNA polymerase Ill-type enzyme from a thermophilic bacterium comprising the steps of: (a) forming a genomic library from the bacterium; .

(b) transforming or transfecting an appropriate host cell with the library of step

(a);

(c) contacting DNA from the transformed or transfected host cell with a DNA probe which hybridizes to a DNA fragment selected from the group consisting of the DNA fragments defined in SEQ ID NO: 6 and the DNA fragments defined in SEQ ID

NO: 8 or the oligonucleotides set forth above; wherein hybridization is conduction under the following conditions: i) hybridization: 1% crystalline BSA (fraction V) (Sigma), 1 mM EDTA,

0.5 M NaHP04 (pH 7.2), 7% SDS at 65 °C for 12 hours and; ii) wash: 5 x 20 minutes with wash buffer consisting of 0.5% BSA, fraction V), ImM Na2EDTA, 40 mM NaHP04 (pH 7.2), and 5% SDS;

(d) assaying the transformed or transfected cell of step (c) which hybridizes to the DNA probe for DNA polymerase Ill-type activity; and (e) isolating a target DNA fragment which codes for the thermostable DNA polymerase Ill-type enzyme.

Also, antibodies including both polyclonal and monoclonal antibodies, and the DNA Polymerase Ill-like enzyme complex and/or their γ and τ subunits or α subunit may be used in the preparation of the enzymes of the present invention as well as other enzymes of similar thermophilic origin. For example, the DNA Polymerase Ill-type complex or its subunits may be used to produce both polyclonal and monoclonal antibodies to themselves in a variety of cellular media, by known techniques such as the hybridoma technique utilizing, for example, fused mouse spleen lymphocytes and myeloma cells.

The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal, antibody-producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA. or transfection with Epstein-Barr virus. See, e.g., Mi Schreier et al., "Hybridoma Techniques" (1980); Hammerling et al., "Monoclonal Antibodies And T- cell Hybridomas" (1 81); Kennett et al.. "Monoclonal Antibodies" (1980); see also U.S. Patent Nos. 4.341,761 : 4,399,121 ; 4.427,783; 4,444,887; 4,451,570; 4,466.917; 4.472,500; 4,491.632: 4,493.890.

Methods for producing polyclonal anti-polypeptide antibodies are well-known in the art. See U.S. Patent No. 4,493,795 to Nestor et al. A monoclonal antibody, typically containing Fab and/or F(ab')₂ portions of useful antibody molecules, can be prepared using the hybridoma technology described in Antibodies - A Laboratory Manual, Harlow and Lane, eds.. Cold Spring Harbor Laboratory, New York (1988), which is incoφorated herein by reference. Briefly, to form the hybridoma from which the monoclonal antibody composition is produced, a myeloma or other self-peφetuating cell line is fused with lymphocytes obtained from the spleen of a mammal hyperimmunized with an elastin-binding portion thereof. A monoclonal antibody useful in practicing the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate antigen specificity. The culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium. The antibody- containing medium is then collected. The antibody molecules can then be further isolated by well-known techniques.

Media useful for the preparation of these compositions are both well-known in the art and commercially available and include synthetic culture media, inbred mice and the like. An exemplary synthetic medium is Dulbecco's minimal essential medium

(DMEM; Dulbecco et al., Virol. 8:396 (1959)) supplemented with 4.5 gm/1 glucose. 20 mm glutamine, and 20% fetal calf serum. An exemplary inbred mouse strain is the Balb/c.

Another feature of this invention is the expression of the DNA sequences disclosed herein. As is well known in the art, DNA sequences may be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate unicellular host.

Such operative linking of a DNA sequence of this invention to an expression control sequence, of course, includes, if not already part of the DNA sequence, the provision of an initiation codon, ATG, in the correct reading frame upstream of the DNA sequence.

A wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids col Εl, pCRl, pBR322, pMB9 and their derivatives, plasmids such as RP4; phage DNAS, e.g., the numerous derivatives of phage λ, e.g., NM989, and other phage DNA, e.g., Ml 3 and filamentous single stranded phage DNA; yeast plasmids such as the 2μ plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs. such as plasmids that have been modified to employ phage DNA or other expression control sequences: and the like.

Any of a wide variety of expression control sequences ~ sequences that control the expression of a DNA sequence operatively linked to it ~ may be used in these vectors to express the DNA sequences of this invention. Such useful expression control sequences include, for example, the early or late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the LTR system, the major operator and promoter regions of phage λ, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), the promoters of the yeast α-mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

A wide variety of unicellular host cells are also useful in expressing the DNA sequences of this invention. These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO, Rl.l, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g., Sf9), and human cells and plant cells in tissue culture.

It will be understood that not all vectors, expression control sequences and hosts will function equally well to express the DNA sequences of this invention. Neither will all hosts function equally well with the same expression system. However, one skilled in the art will be able to select the proper vectors, expression control sequences, and hosts without undue experimentation to accomplish the desired expression without departing from the scope of this invention. For example, in selecting a vector, the host must be considered because the vector must function in it. The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, will also be considered.

In selecting an expression control sequence, a variety of factors will normally be considered. These include, for example, the relative strength of the system, its controllability, and its compatibility with the particular DNA sequence or gene to be expressed, particularly with regard to potential secondary structures. Suitable unicellular hosts will be selected by consideration of, e.g., their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements, as well as the toxicity to the host of the product encoded by the DNA sequences to be expressed, and the ease of purification of the expression products.

Considering these and other factors a person skilled in the art will be able to construct a variety of vector/expression control sequence/host combinations that will express the DNA sequences of this invention on fermentation or in large scale animal culture.

It is further intended that analogs may be prepared from nucleotide sequences of the protein complex/subunit derived within the scope of the present invention. Analogs, such as fragments, may be produced, for example, by pepsin digestion of bacterial material. Other analogs, such as muteins. can be produced by standard site-directed mutagenesis oϊdnaX. dnaE, dnaQ or dnaN coding sequences. Especially useful may be a mutation in dnaE that provides the polymerase with the ability to incoφorate all four ddNTPs with equal efficiency thereby producing an even binding pattern in sequencing gels, as discussed above and with reference to Tabor et al. 1995, supra.. As mentioned above, a DNA sequence corresponding to dnaX, dnaQ, dnaE or dnaN, or encoding the subunits of the DNA Polymerase III of the invention can be prepared synthetically rather than cloned. The DNA sequence can be designed with the appropriate codons for the amino acid sequence of the subunit(s) of interest. In general, one will select preferred codons for the intended host if the sequence will be used for expression. The complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge, Nature, 292:756 (1981); Nambair et al., Science, 223:1299 (1984); Jay et al., J. Biol. Chem., 259:6311 (1984).

Synthetic DNA sequences allow convenient construction of genes which will express DNA Polymerase III analogs or "muteins". Alternatively, DNA encoding muteins can be made by site-directed mutagenesis of native dnaX. dnaQ, dnaE or dnaN genes or their corresponding cDNAs. and muteins can be made directly using conventional polypeptide synthesis.

A general method for site-specific incorporation of unnatural amino acids into proteins is described in Christopher J. Noren, Spencer J. Anthony-Cahill, Michael C. Griffith. Peter G. Schultz, Science, 244: 182-188 (April 1989). This method may be used to create analogs with unnatural amino acids.

GENERAL DESCRIPTION

As discussed above, the present invention has as one of its characterizing features, that a Polymerase Ill-type enzyme as defined hereinabove, has been discovered in a hermophile, that has the structure and function of a chromosomal replicase. This structure and function confers significant benefit when the enzyme is employed in procedures such as PCR where speed and accuracy of DNA reconstruction is crucial. Chromosomal replicases are composed of several subunits in all organisms (Kornberg and Baker. 1992). In keeping with the need to replicate long chromosomes, replicases are rapid and highly processive multiprotein machines. All cellular replicases examined to date derive their processivity from one subunit that is shaped like a ring and completely encircles DNA (Kuriyan and O'Donnell, 1993; Kelman and O'Donnell, 1994). This "sliding clamp" subunit acts as a mobile tether for the polymerase machine (Stukenberg et. al.. 1991). The sliding clamp does not assemble onto the DNA by itself, but requires a complex of several proteins, called a "clamp loader" which couples ATP hydrolysis to the assembly of sliding clamps onto DNA (O'Donnell et. al., 1992). Hence, Pol Ill-type cellular replicases are comprised of three components: a clamp, a clamp loader, and the DNA polymerase.

An overall goal is to identify and isolate all of the genes encoding the replicase subunits from a thermophile for expression and purification in large quantity. Following this, the replication apparatus can be reassembled from individual subunit components for use in kits, PCR, sequencing and diagnostic applications (Onrust et. al, 1995).

As a beginning to identify and characterize the replicase of a thermophile. we started by looking for a homologue to the prokaryotic dnaX gene which encode subunits (γ and τ) of the replicase. The dnaX gene has another homologue, holB, which encodes yet another subunit (δ') of the replicase. The amino acid sequence of δ' (encoded by holA) and τ/γ subunits (encoded by dnaX) are particularly highly conserved in evolution from prokaryotes to eukaryotes ( Chen et. al., 1992; O'Donnell et. al., 1993; Onrust et. al., 1993: Carter et. al., 1993; Cullman et. al., 1995).

The organism chosen for study and exposition herein is the exemplary extreme thermophile, Thermus thermophilus (T.th.). It is understood that other members of the class such as the eubacterium Thermatoga are expected to be analogous in both structure and function. Thus, the investigation of T.th. proceeded and initially, a T.th. homologue of dnaX was identified. The gene encodes a full length protein of 529 amino acids. The amino terminal third of the sequence shares over 50% homology to dnαX genes as divergent as E. coli (gram negative) and B. subtilis (gram positive). The T.th. dnαX gene contains a DNA sequence that provides a translational frameshift signal for production of two proteins from the same gene. Such frameshifting has been documented only in the case of E. coli (Tsuchihashi and Kornberg, 1990; Flower and McHenry, 1990: Blinkowa and Walker, 1990). No frameshifting has been documented to occur in the όfaαA^'homologues (RFC subunit genes) of yeast and humans (Eukaryotic kingdom).

The presence of a dnαX gene that produces two subunits implies that T. th. has a clamp loader (γ) and is organized by τ into a three component Pol Ill-type replicase. The three components of its replicase may be organized into a holoenzyme particle like the replicative DNA polymerase of Escherichia coli, DNA polymerase III holoenzyme. The E. coli DNA polymerase III holoenzyme contains 10 different subunits, some in copies of two or more for a total composition of 18 polypeptide chains (Baker and Kornberg, 1992; Onrust et. al., 1995). The holoenzyme is composed of three major activities: the 3-subunit DNA polymerase core (αeθ), the β subunit DNA sliding clamp, and the 5-subunit γ complex clamp loader (γδδ'χψ). This 3 component strategy generalizes to eukaryotes which utilize a clamp (PCNA) and a 5-subunit RFC clamp loader (RFC) which provide processivity to DNA polymerase δ (reviewed in Kelman and O'Donnell, 1994).

In E. coli, the three components are organized into one holoenzyme particle by the τ subunit, that acts as a "glue" protein (Onrust and O'Donnell, 1995). One dimer of τ holds together two core polymerases into one particle which are utilized for the coordinated and simultaneous replication of both strands of duplex DNA (McHenry, 1982; Maki et. al., 1988; Yuzhakov et. al., 1996). The "glue" protein τ subunit also binds one clamp loader (called γ complex) thereby acting as a scaffold for a large superstructure assembly called DNA polymerase III holoenzyme. The gene encoding τ. called dnaX. also encodes the γ subunit of DNA polymerase III. The β subunit then associates with Pol III to form the DNA polymerase III holoenzyme. The γ subunit is approximately 2/3 the length of τ. γ shares the N-terminus of τ, but is truncated by a translational frameshifting mechanism that, after the shift, encounters a stop codon within two amino acids (Tsuchihashi and Kornberg, 1990; Flower and McHenry, 1990; Blinkowa and Walker. 1990). Hence, γ is the N-terminal 453 amino acids of τ, but contains one unique residue at the C-terminus (the penultimate codon encodes a Lys residue which is the same sequence as if the frameshift did not take place). This frameshift is highly efficient and occurs approximately 50% of the time.

The sequence of the γ and τ subunits encoded by the dnaX gene are homologous to the clamp loading subunits in all other organisms extending from gram negative bacteria through gram positive bacteria, the Archeae Kingdom and the Eukaryotic Kingdom from yeast to humans (O'Donnell et al., 1993). All of these organisms utilize a three component replicase (DNA polymerase, clamp and clamp loader) and in these cases the 3 components appear to behave as independent units in solution rather than forming a large holoenzyme superstructure. For example, in eukaryotes from yeast to humans, the clamp loader is the five subunit RFC, the clamp is PCNA and the polymerases δ and e are all stimulated by the PCNA clamp assembled onto primed DNA by RFC (reviewed in Kelman et. al., 1994).

The discovery of a dnaX gene in T.th. provided confidence that thermophilic bacteria would contain a three component Pol Ill-type enzyme. Hence, we proceeded to identify the dnaQ and dnaN genes encoding, respectively, the proofreading 3'-5' exonuclease, and the β DNA sliding clamp subunits of a Pol III -type enzyme. Following this, we purified from extracts of T.th. cells, a Pol Ill-type enzyme. This enzyme preparation had the unique property of extending a single primer around a long 7.2 kb single strand DNA genome of M13mpl 8 bacteriophage. Such a primer extension assay serves as a tool to detect and identify the Pol Ill-type of enzyme in cell extracts. The enzyme was confirmed to be a Pol Ill-type enzyme based on its reactivity with antibody directed against the E. coli a subunit (the DNA polymerase subunit) and antibody directed against E. coli γ subunit. Proteins corresponding to α. τ, γ, δ and δ' were easily visible and will lend themselves to identification of the genes through use of peptide microsequencing followed by primer design for PCR amplification. From this DNA pol Ill-type preparation we obtained peptide sequence of the α subunit enabling us to obtain the dnaE gene encoding the α subunit (DNA polymerase) of the Pol Ill-type enzyme.

These methods should be widely applicable to other thermophilic bacteria. Additional antibody reaents against other Pol Ill-type enzyme components, such as RFC subunits, DNA polymerase delta, epsilon or beta, and the PCNA clamp from known organisms can be made quite easily as polyclonal or monoclonal antibody preparations using as antigen either naturally purified sequence, recombinant sequence, or synthetic peptide sequence. Examples of known sequences of these Pol Ill-type enzymes are to be found in: 1) DNA polymerases (Braithwaite and Ito, 1993), RFC clamp loaders (Cullman et. Al., 1995), and PCNA (Ielman and O'Donnell, 1995).

Braithwaite, D.K. and Ito, J. (1993) Compilation, alignment, and phylogenetic relationships of DNA polymerases. Nuc. Acids Res. 21, 787-802.

Cullman G., Fein, K., Kobayashi, R., and Stillman. B. (1995) Characterization of the five replication factor C genes of Saccharomyces cerevisiae. Mol. Cell. Biol. 15, 4661-4671.

Kelman, Z., and O'Donnell, M. (1995) Structural and functional similarities of prokaryotic and eukaryotic DNA polymerase sliding clamps. Nucl Acids Res. 23, 3613-3620. The remaining genes of Pol III needed for efficient extension of primed templates should be easy to obtain from the T.th. Pol III by similar methods as those described herein. These genes will provide the subunit preparations through use of standard recombinant techniques and protein purification protocols. The protein subunits can then be used to reconstitue the enzyme complexes as they exist in the cell. This type of reconstitution of Pol III has been demonstrated using the protein subunits of DNA polymerase III holoenzyme from E. coli to assemble the entire particle. See e.g., U.S. Patent No. 5,583,026. issued December, 1996, O'Donnell, M.Ε.; and U.S. Patent No. 5,668,004, issued September, 1997, both to one of the inventors herein, and Onrust et. al. 1995b. The disclosures of these references are incoφorated herein in their entireties.

The following experiments illustrate the identification and characterization of the enzymes and constructs of the present invention. Accordingly, in Examples 1-8 below, the identification and expression of the γ and τ is presented, as the first step in the elucidation of the Polymerase III reflective of the present invention. Examples 9- 13 which follow set forth the protocol for the purification of the remainder of the subunits of the enzyme that represent substantial entirety of the functional replicative machinery of the enzyme.

EXAMPLE 1

EXPERIMENTAL PROCEDURES

Materials - DNA modification enzymes were from New England Biolabs. Labelled nucleotides were from Amersham, and unlabeled nucleotides were from New England Biolabs The Alter- 1 vector was from Promega. pET plasmids and E. coli strains, BL21(DE3) and BL21(DE3)pLysS, were from Novagen. Oligonucleotides were from Operon. Buffer A is 20mM Tris-HC 1 (pH 7.5), 0.1 mM EDTA, 5mMDTT, and 10% glycerol. Genomic DNA

Thermus thermophilus (strain HB8) was obtained from the American Type Tissue Collection. Genomic DNA was prepared from cells grown in 0.1 1 of (Thermus medium N697 (ATCC: 4 γ yeast extract. 8.0 g polypeptone (BBL 1 1910), 2.0 g NaCl, 30.0 g agar. 1.0 L distilled water) at 75 °C overnight. Cells were collected by centrifugation at 4°C and the cell pellet was resuspended in 25 ml of 100 mM Tris-HCl (pH 8.0), 0.05 M EDTA, 2 mg/ml lysozyme and incubated at room temperature for 10 min. Then 25 ml 0.10 M EDTA (pH 8.0), 6% SDS was added and mixed followed by 60 ml of phenol. The mixture was shaken for 40 min. followed by centrifugation at 10,000 X G for 10 min. at room temperature. The upper phase (50 ml) was removed and mixed with 50 ml. of phenol xhloroform (50:50 v/v) for 30 min. followed by centrifugation for 10 min. at room temperature. The upper phase was decanted and the DNA was precipitated upon addition of 1/10th volume 3 M sodium acetate (pH 6.5) and 1 volume ethanol. The precipitate was collected by centrifugation and washed twice with 2 ml of 80% ethanol, dried and resuspended in 1 ml T.E. buffer (lOmM Tris Hcl (pH 7.5), ImM EDTA). .

Cloning of dnaX - DNA oligonucleotides for amplification of T.th. genomic DNA were as follows. The upstream 32mer (5'-CGCAAGCTICACGCSTACCTSTTCTCCGGSAC-3') (S indicates a mixture of G and C) consists of a Hind III site within the first 9 nucleotides (underlined) followed by codons encoding the following sequence (HAYLFSGT). The downstream 34 mer (5'-CGCG ATTC_GTGCTCSGGSGGCTCCTCSAGSGTC-3') consists of an EcoRI site (underlined) followed by codons encoding the sequence KTLEEPPEH on the complementary strand. The amplification reactions contained 10 ng T.th. genomic DNA. 0.5 mM of each primer, in a volume of 100 μl of Vent polymerase reaction mixture according to the manufacturers instructions (10 μl ThermoPol Buffer, 0.5 mM each dNTP and 0.5 mM MgS0₄). Amplification was performed using the following cycling scheme: 5 cycles of: 30 s at 95.5 °C, 30 s at 40°C, 2 min. at 72°C; 5 cycles of: 30 s at 95.5°C, 30 s at 45°C, and 2 min. at 72°C; and 30 cycles of: 30 s at 95.5°C. 30 s at 50°C, and 30 s at 72°C. Products were visualized in a 1.5 % native agarose gel.

Genomic DNA was digested with either Xhol, Xbal, StuI, PstI, Ncol, Mlul, Kpnl, Hindlll, EcoRI, Eagl. Bgll, or BamHI, followed by Southern analysis in a native agarose gel (Maniatis et. al., 1982). Approximately 0.5 μg of digest was analyzed in each lane of a 0.8 % native agarose gel followed by transfer to an MSI filter (Micron Separations Inc.). The transfer included the following steps:

1. The agarose gel was soaked in 500 ml of 1% HCl with gentle shaking for 10 min.

2. Then the gel was soaked in 500 ml of 0.5 M NaOH + 1.5 M NaCl for 40 min. 3. After that the gel was soaked in 500 ml of 1M ammonium acetate for 1 h.

4. The DNA was transferred to the MSI filter with the use of blotting paper for 4 h.

5. The filter was kept at 80 °C for 15 min. in the oven.

6. The pre-hybridization step was run in 10 ml of Hybridization solution (1% crystalline BSA (fraction V) (Sigma), 1 mM EDTA. 0.5 M NaHP04 (pH 7.2), 7% SDS) at 65 °C for 30 min.

7. The probe, radiolabelled by the random priming method (see below), was added to the pre-hybridization solution and kept at 65 °C for 12 h.

8. The filter was washed with low stringency with 200 ml of the wash buffer (0.5% BSA, fractionV). ImM Na2EDTA, 40 mM NaHP04 (pH 7.2), 5% SDS with gentle shaking for 20 min. This step was repeated 5 times, followed by exposure to X-ray film (XAR-5, Kodak).

As a probe, the PCR product was radiolabelled by random as follows.

1. 14 ml of the mixture containing 0.2 μg of PCR product DNA, 1 μg of the pd(N6) (Promega) and 2.5 ml of the 10X Klenow reaction buffer (100 mM Tris-HCl (pH 7.5), 50 mM MgCl₂, 75 mM dithiothreitol) were boiled for 10 min. and then kept at 4°C .

2. The reaction volume was increased up to 25 μl, containing in addition 33 μM of each dNTP, except dATP, 10 μCi [α-³²P] dATP (800 Ci/mM), and 2 units of Klenow enzyme. The reaction mixture was incubated 1.5 h. 3. 2 mg of sonicated herring sperm DNA (GibcoBRL) was added to the reaction and the volume was increased to 2 ml using hybridization solution. The sample was then boiled for 10 min.

A genomic library of Xbal digested DNA was prepared upon treating 1 μg genomic T.th. DNA with 10 units of Xbal in 100 μl of NEBuffer N2 (50 mM NaCl, 10 mM Tris-HCl (pH 7.9). 10 mM MgC12, 1 mM DTT) for 2 h at 37°C. The digested DNA was purified by phenol chloroform extraction and ethanol precipitation. The Alter- 1 vector (0.5 μg)(Promega) was digested with 1 unit of Xbal in NEBuffer N2 and then purified by phenol/chloroform extraction and ethanol precipitation. One microgram of genomic digest was incubated with 0.05 μg of digested Alter- 1 and 20 U of T4 ligase in 30 μl of ligase buffer (50 mM Tris-HCl (pH 7.8), 10 mM MgC12, 10 mM DTT and 1 mM ATP) at 15 °C for 12 h. The ligation reaction was transformed into the DH5α strain of E. coli and transformants were plated on LB plates containing ampicillin and screened for the dnaX insert using the radiolabelled PCR probe as follows:

1. The colonies tested were lifted onto MSI filters, approximately 100 colonies to each filter.

2. The filters, removed from the LB/Tc plates, were placed side up on a sheet of Whatman 3 MM paper soaked with 0.5 M NaOH for 5 min. 3. The filters were transferred to a sheet of paper soaked with 1 M Tris-HCl (pH 7.5) for 5 min.

4. The filters were placed on a sheet of paper soaked in 0.5 M Tris-HCl (pH 7.5), 1.25 M NaCl for 5 min.

5. After drying by air, the filters were heated in the oven 80 °C for 15 min. and then were analyzed by Southern hybridization.

Plasmid DNA was prepared from 20 positive colonies; of these 6 contained the expected 4 kb insert when digested with Xbal. Sequencing of the insert was performed by the Sanger method using the Vent polymerase sequencing kit according to the manufacturers instructions (New England Biolabs).

Identification of the dnaX gene

The dnaX genes of the gram negative. E. coli, and the gram positive B. subtilis, share more than 50% identity in amino acid sequence within the N-terminal 180 residues containing the ATP-binding domain (Fig. 2). Two highly conserved regions (shown in bold in Fig. 2) were used to design oligonucleotide primers for application of the polymerase chain reaction to T.th. genomic DNA. The expected PCR product, including the restriction sites (i.e. before cutting) is 345 nucleotides. Use of these primers with genomic T.th. DNA resulted in a product of the expected size. The PCR product was then radiolabelled and used to probe genomic DNA in a Southern analysis (Fig. 3). Genomic DNA was digested with several different restriction endonucleases, electrophoresed in a native agarose gel and then probed with the PCR fragment. The Southern analysis showed an Xbal fragment of approximately 4 kb, more than sufficient length to encode the dnaX gene. Other, restriction nucleases produced fragments that were significantly longer, or produced two or more fragments indicating presence of a site within the coding sequence of dnaX.

To obtain full length dnaX, genomic DNA was digested with Xbal and ligated into Xbal digested Alter- 1 vector. Ligated DNA was transformed into DH5 alpha cells, and colonies were screened with the labeled PCR probe. Plasmid DNA was prepared from 20 positive colonies and analyzed for the appropriate sized insert using Xbal. Six of the twenty clones contained the expected 4 kb Xbal fragment as an insert, the sequence of which is shown in Figs. 4A and 4B.

The frameshift site

The dnaX gene of E. coli produces two proteins, the γ and τ subunits, by a -1 frameshift (Tsuchihashi and Kornberg, 1990; Flower and McHenry, 1990; Blinkowa and Walker, 1990). The full length product yields τ. and the frameshift results in addition of one amino acid before encountering a stop codon to produce γ. The -1 frameshift site in the E. coli dnaX gene contains the sequence, A AAA AAG, which follows the X XXY YYZ rule found in retroviral genes (Jacks et. al., 1988). This "slippery sequence" preserves the initial two residues of the tRNAs in the aminoacyl and peptidyl sites both before and after the frameshift. Mutagenesis of the E. coli dnaX frameshifting site has shown that the first three residues can be nucleotides other than A, but that A's in the second set of three nucleotides is important to frameshifting (Tsuchihashi and Brown, 1992).

Immediately downstream of the stop codon is a potential stem-loop structure which enhances frameshifting, presumably by causing the ribosome to pause. Further, the AAG codon lacks a cognate tRNA in E. coli and thus the G residue may facilitate the pause, and has been shown to aid the vigorous frameshifting observed in the E. coli dnaX gene (Tsuchihashi and Brown, 1992). A fourth component of frameshifting in the E. coli dnaX gene is presence of an upstream Shine-Dalgarno sequence which is thought to pair with the 16S rRNA to increase the frequency of frameshifting still further (Larsen et. al.. 1994).

Examination of the T.th. dnaX sequence reveals a single site that fulfills the X XXY YYZ rule in which positions 4-7 are A residues. The site is unique from that in E. coli as all seven residues are A, and the heptanucleotide sequence is flanked by another A residue on each side (i.e. A9). Surprisingly, the stop codon immediately downstream of this site is in the -2 frame, although there is a stop codon in the -1 frame 28 nucleotides downstream of the -2 stop codon. Indeed, a -2 frameshift would fulfill the requirement that the first two nucleotides of each codon in the peptidyl and aminoacyl sites be conserved during either a -1 or a -2 frameshift. As with the case of E. coli dnaX, there are secondary structure step loop structures immediately downstream. Finally, there is a Shine-Dalgarno sequence immediately adjacent to the frameshift site, as well as another Shine-Dalgarno sequence 22 nucleotides upstream of the frameshift site. Assuming the first stop codon is utilized (i.e. -2 frameshift), the predicted size of the γ subunit in T.th. is 454 amino acids for a mass of 49.8 kDa, over 2 kDa larger than the 431 residue γ subunit (47.5 kDa) of E. coli. This would result in 2 residues after the -2 frameshift (i.e. after the GluLysLys. the residues LysAla would be added) to be compared to the result of the - 1 frameshift in E. coli which also results in 2 residues (LysGlu). In the event that a -1 frameshift were utilized in the T.th. dnaX gene, then an additional 12 residues would be added following the frameshift for a molecular mass of 50.8 kDa (i.e. after the GluLysLys, the residues LysProAspProLysAlaProProGlyProThrSer would be added). As explained later, this nucleotide sequence was found to promote both -1 and -2 frameshifting in E. coli (Fig. 8). But first, we examined T.th. cells by Western analysis for the presence of two subunits homologous to E. coli γ and τ.

EXAMPLE 2

Frameshifting analysis of the T.th. dn X gene Frameshifting was analyzed by inserting the frameshift site into lacZ in the three different reading frames, followed by plating on X-gal and scoring for blue or white colony formation (Weiss et. al.. 1987). The frameshifting region within T.th dnaX was subcloned into the EcoRI/BamHI sites of pUC 19. These sites are within the polylinker inside of the β-galactosidase gene. Three constructs were produced such that the insert was either in frame with the downstream coding sequence of β-galactosidase, or were out of frame (either -1 or -2). An additional three constructs were designed by mutating the frameshift sequence and then placing this insert into the three reading frames of the β-galactosidase gene. These six plasmids were constructed as described below.

The upstream primer for the shifty sequences was 5'-gcg egg ate egg agg gag aaa aaa aaa gec tea gec ca-3'. The BamHI site for cloning into pUC is underlined. Also, the stop codon. tga, has been mutated to tea (also underlined). The upstream primer for the mutant shifty sequence was: 5'-gcg egg ate egg agg gag aga aga aaa gec tea gec ca-3'. The mutant sequence contains two substitutions of a G for an A residue in the polyA stretch (underlined). Three downstream primers were utilized with each upstream primer to create two sets of three inserts in the 0 frame, -1 frame and -2 frame. The sequence of these primers, and the length of insert (after cutting with EcoRI and BanHI and inserting into pUC 19) are as follows: 5'-gaa tta aat teg cgc ttc ggg agg tgg g-3' (0 frameshift. total 58 nucleotide insert); 5'-gcg cga att cgc get teg gga ggt ggg-3' (-1 frame, 54mer insert); and 5'-gcg cga att egg gcg ctt cag gag gtg gg-3' (-2 frame, 56mer insert). The downstream primers have an EcoRI site (underlined); the EcoRI site of the 0 frame insert was blunt ended to produce the greater length insert (converting the EcoRI site to an aattaatt sequence). Also, the teg sequence, which produces the tga stop codon (underlined) was mutated to tea in the -2 downstream primer so that readthrough would be allowed after the frameshift occurred.

In summary, a region surrounding the frameshift site and ending at least 5 nucleotides past the -1 frameshift stop codon was inserted into the β galactosidase gene of pUC19 in the three different reading frames (stop codons were mutated to prevent stoppage following a frameshift). These three plasmids were introduced into E. coli and plated with X-gal. The results, in Fig. 8, show that blue colonies were observed after 24 h incubation with all three plasmids and therefore both -1 and -2 frameshifting had occurred.

To further these results, two γ residues were introduced into the polyA tract which should disrupt the ability of this sequence to direct frameshifts. The mutated slippery sequence was inserted into pUC 19 followed by transformation into E. coli and plating on X-gal. The results showed that both -1 and -2 frameshifting was prevented, further supporting the fact that frameshifting requires the polyA tract as expected (Fig. 8). 8/45452

65 EXAMPLE 3

Expression vector for T.th. γ and τ

The dnaX gene was cloned into the pET16 expression vector in the steps shown in

Fig. 9. First, the bulk of the gene was cloned into pET16 by removing the Pmll/Xbal fragment from pAlterdnaX, and placing it into Smal/Xbal digested Pucl9 to yield Pucl9dnaXCterm. The N-terminal sequence of the dnaX gene was then reconstructed to position an Ndel site at the N-terminus. This was performed by amplifying the 5' region encoding the N-terminal section of γ/τ using an upstream primer containing an Ndel site that hybridizes to the dnaX gene at the initiating gtg codon (i.e. to encode Met where the Met is created by the PCR primer, and the Val is the initiating gtg start codon of dnaX). The primer sequence for this 5' end was: 5'-gtggtgcatatg gtg age gec etc tac cgc c-3' (where the Ndel site is underlined, and the coding sequence of dnaX follows). The downstream primer hybridizes past the Pmll site at nucleotide positions 987 - 1004 downstream of the initiating gtg (primer sequence: 5'-gtggtggtcgac cca gga ggg cca cct cca g-3' where the initial 12 nucleotides contain a SalGI restriction site, followed by the sequence from the region downstream the stop codon). The 1.1 kb nucleotide PCR product was digested with Pmll Ndel and the Pmll/Ndel fragment was ligated into Ndel/Pmll digested Puc 19dnaXCterm to form Pucl9dnaX. The Pucl9dnaX plasmid was then digested with Ndel and Sail and the 1.9 kb fragment containing the dnaX gene was purified using the Sephaglas BandPrep Kit

(Pharmacia-LKB). pETlόb was digested with Ndel and Xhol. Then the full length dnaX gene was ligated into the digested pETlόb to form pETdnaX.

EXAMPLE 4 - Expression of T.th. v and τ

As discussed in the previous example, the dnaX gene was engineered into the T7 based IPTG inducible pET16 vector such that the initiation codon was placed precisely following the Met residue N-terminal leader sequence (Fig. 9). This should produce a protein containing the entire sequence of γ and τ, along with a 21 residue leader containing 10 contiguous His residues (tagged-τ = 60.6 kDa: tagged-γ = 52.4 kDa for -2 frameshift). The pETdnaX plasmid was introduced into BL21(DE3)pLysS cells harboring the gene encoding T7 RNA polymerase under control of the lac repressor. Log phase cells were induced with IPTG and analyzed before and after induction in an SDS polyacrylamide gel (Fig. 10, lanes 1 and 2). The result shows that upon induction, two new proteins are expressed with the approximate sizes expected of the T.th. γ and τ subunits (larger than E. coli γ, and smaller than E. coli τ). The two proteins are produced in nearly equal amounts, similar to the case of the E. coli γ and τ subunits. Western analysis using antibodies against the E. coli γ and τ subunits cross reacted with the induced proteins further supporting their identity as T.th. γ and τ (data not shown, but repeated with the pure subunits shown in Fig. 10, lane 6).

EXAMPLE 5 Purification of T. th. γ and τ The His-tagged T.th. γ and τ proteins were purified from 6 L of induced E. coli cells containing the pET dnaX plasmid. Cells were lysed. clarified from cell debris by centrifugation and the supernatant was applied to a HiTrap chelate affinity column. Elution of the chelate affinity column yielded approximately 35 mg of protein in which the two predominant bands migrated in a region consistent with the molecular weight predicted from the dnaX gene (Fig. 10, lane 3), and produced a positive signal by Western analysis using polyclonal antibody directed against the E. coli γ and τ subunits (lane 4). The γ and τ subunits are present in nearly equal amounts consistent with the nearly equal expression of these proteins in E. coli cells harboring the pETdnaX plasmid.

The γ and τ subunits were further purified by gel filtration on a Superose 12 column (Fig. 10, lane 4; Fig. 1 1). Recovery of T.th. γ and τ subunits through gel filtration was 81%. The E. coli γ and τ subunits. when separated from one another, elute during gel filtration as tetramers. A mixture of E. coli γ/τ results in a mixed tetramer of γ2t2 along with τ4 and γ4 tetramers (Onrust et. al.. 1995). The mixture of T.th. γ/τ elutes ahead of the 150 kDa marker, and thus is consistent with the expected mass of a γ2t2 tetramer (225 kDa) and τ4 and γ4 tetramers.

As described earlier, the dnaX frameshifting sequence could produce either a -1 or -2 framehift to yield a His-tagged γ subunit of mass either 53.3 kDa or 52.4 kDa, respectively. The difference in these two possible products is too close to determine from migration in SDS gels. It also remains possible that two γ products are present and do not resolve under the conditions used. The exact protocol for this purification is described below.

Six liters of BL21(DE3)pLysSpETdnaX cells were grown in LB media containing 50 μg/ml ampicillin and 25 μg/ml chloramphenicol at 37°C to an O.D. of 0.8 and then IPTG was added to a concentration of 2 mM. After a further 2 h at 37°C. cells were harvested by centrifugation and stored at -70 °C. The following steps were performed at 4°C. Cells (15 g wet weight) were thawed and resuspended in 45 ml IX binding buffer (5 mM imidizole, 0.5 M NaCl, 20 mM Tris HCl (final pH 7.5)) using a dounce homogenizer to complete cell lysis and 450 ml of 5% polyamine P (Sigma) was added. Cell debris was removed by centrifugation at 18,000 φm for 30 min. in a Sorvall SS24 rotor at 4°C. The supernatant (Fraction I, 40 ml, 376 mg protein) was applied to a 5 ml HiTrap Chelating Separose column (Pharmacia-LKB). The column was washed with 25 ml of binding buffer, then with 30 ml of binding buffer containing 60 mM imidizole, and then eluted with 30 ml of 0.5 M imidizole, 0.5 M NaCl, 20 mM Tris-HCl (pH 7.5). Fractions of 1 ml were collected and analyzed on an 8% Coomassie Blue stained SDS polyacrylamide gel. Fractions containing subunits migrating at the T.th γ and τ positions, and exhibiting cross reactivity with antibody to E. coli γ and τ in a Western analysis, were pooled and dialyzed against buffer A (20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 5 mM DTT and 10% glycerol) containing 0.5 M NaCl (Fraction II, 36 mg in 7 ml). Fraction II was diluted 2-fold with buffer A and passed through a 2 ml ATP agarose column equilibrated in buffer A containing 0.2 M NaCl to remove any E. coli γ complex contaminant. Then 0.18 mg (300 ml) Fraction II was gel filtered on a 24 ml Superose 12 column (Pharmacia-LKB) in buffer A containing 0.5 M NaCl. After the first 216 drops, fractions of 200 μl were collected (Fraction III) and analyzed by Western analysis (by procedures similar to those described in Example 6). by ATPase assays and by Coomassie Blue staining of an 8% Coomassie Blue stained SDS polyacrylamide gel. The Coomassie stained gels and Western analysis of recombinant T.th. gamma and tau for these purification steps are summarized in Fig. 10.

EXAMPLE 6

Western Analysis of T.th. cells for presence of γ and τ subunits

Polyclonal antibody to E. coli γ/τ - E. coli γ subunit was prepared as described ( Studwell-Vaughan and O'Donnell, 1991 ). Pure γ subunit (100 μg) was brought up in Freund's adjuvant and injected subcutaneously into a New Zealand Rabbit (Poccono Rabbit Farms). After two weeks, a booster consisting of 50 μg γ in Freund's adjuvant was administered, followed after two weeks by a third injection (50 μg).

The homology between the amino terminal regions of T.th. and E. coli γ/τ subunits suggested that there may be some epitopes in common between them. Hence, polyclonal antibody directed against the E. coli γ/τ subunits was raised in rabbits for use in probing T.th. cells by Western analysis. Fig. 7 shows the results of a Western analysis of whole T.th. cells lysed in SDS. The results show that in T.th. cells, the antibody is rather specific for two high molecular proteins which migrate in the vicinity of the molecular masses of E. coli γ and τ subunits.

Procedure for Western Analysis Samples were analyzed in duplicate 10 % SDS polyacrylamide gels by the Western method (Towbin et. al. 1979). One gel was Coomassie stained to evaluate the pattern of proteins present, and the other gel was then electroblotted onto a nitrocellulose membrane (Schleicher and Schuell). For molecular size markers, the kaliedoscope molecular weight markers (Bio-Rad) were used to verify by visualization that transfer of proteins onto the blotted membrane had occurred. The gel used in electroblotting was also stained after electroblotting to confirm that efficient transfer of protein had occured. Membranes were blocked using 5% non-fat milk, washed with 0.05% Tween in TBS (TBS-T) and then incubated for over 1 h with a 1/5000 dilution of rabbit polyclonal antibody directed against E. coli γ and τ in 1 % gelatin in TBS-T at room temperature. Membranes were washed using TBS-T buffer and then antibody was detected on X-ray film (Kodak) by using the ΕCL kit from (Amersham) and the manufactures reccommended procedures.

Samples included: 1 ) a mixture of E. coli γ (15 ng) and τ (15 ng) subunits; 2) T.th. whole cells (100 μl) suspended in cracking buffer; and 3) purified T.th. γ and τ fraction II (0.6 μg as a mixture).

EXAMPLE 7 Characterization of the ATPase Activity of γ/τ - The E. coli τ subunit is a DNA dependent ATPase (Lee and Walker, 1987; Tsuchihashi and Kornberg, 1989). The γ subunit binds ATP but does not hvdrolyze it even in the presence of DNA unless other subunits of the DNA polymerase III holoenzyme are also present (Onrust et. al., 1991). Next we examined the T.th. γ/τ subunits for DNA dependent ATPase activity. The γ/τ preparation was, in fact, a DNA stimulated ATPase (Fig. 1 1, top panel). The specific activity of the T.th. γ/τ was 11.5 mol ATP hydrolyzed mol γ/τ (as monomer and assuming an equal mixture of the two). Furthermore, analysis of the gel filtration column fractions shows that the ATPase activity coelutes with the T.th. g/t subunits. supporting evidence that the weak ATPase activity is intrinsic to the γ/τ subunits (Fig. 1 1). The specific activity of the γ/τ preparation before gel filtration was the same as after gel filtration (within 10%). further indicating that the DNA stimulated ATPase is an inherent activity of the γ/τ subunits. Presumably, only the τ subunit contains ATPase activity, as in the case of E. coli. Assuming only T.th. τ contains ATPase activity, its specific activity is twice the observed rate (after factoring out the weight of γ). This rate is still only one-fifth that of E. coli τ.

The T.th. γ/τ ATPase activity is lower at 37°C than at 65 °C (middle panel), consistent with the expected behavior of protein activity from a thermophilic source. However, there is no apparent increase in activity in proceeding from 50 °C to 65 °C (the rapid breakdown of ATP above 65 °C precluded measurement of ATPase activity at temperatures above 65 °C). In contrast, the E. coli τ subunit lost most of its ATPase activity upon elevating the temperature to 50°C (middle panel). These reactions contain no stabalizers such as a nonionic detergent or gelatin, nor did they include substrates such as ATP, DNA or magnesium.

Last, the relative stability of T.th. γ/τ and E. coli γ/τ to addition of NaCl (Fig. 12, bottom panel) was examined. Whereas the E. coli τ subunit rapidly lost activity at even 0.2 M NaCl. the T.th. γ/τ retained full activity in 1.0 M NaCl and was still 80 % active in 1.5 M NaCl. The detailed procedure for the ATPase activity assay is described below.

ATPase assays: ATPase assays were performed in 20 μl of 20 mM Tris-HCl (pH 7.5). 8 mM MgCL containing 0.72 μg of M13mpl8 ssDNA (where indicated), 100 mM [γ-³²P]-ATP (specific activity of 2000-4000 cpm/pmol), and the indicated protein. Some reactions contained additional NaCl where indicated. Reactions were incubated at the temperatures indicated in the figure legends for 30 min. and then were quenched with an equal volume of 25 mM ΕDTA (final). The aliquots were analyzed by spotting them (1 μl each) onto thin layer chromatography (TLC) sheets coated with Cel-300 polyethyleneimine (Brinkmann Instruments Co.). TLC sheets were developed in 0.5 M lithium chloride, 1 M formic acid. An autoradiogram of the TLC chromatogram was used to visualize Pi at the solvent front and ATP near the origin which were then cut from the TLC sheet and quantitated by liquid scintillation. The extent of ATP hydrolyzed was used to calculate the mol of Pi released per mol of protein per min. One mol of E. coli τ was calculated assuming a mass of 71 kDa per monomer. The T.th. γ and τ preparation was treated as an equal mixture and thus one mole of protein as monomer was the average of the predicted masses of the γ and τ subunits (54 kDa).

EXAMPLE 7

Western analysis of T.th. cells for presence of γ and τ subunits - The homology between the amino terminal regions of T.th. and E. coli γ/τ subunits suggested that there may be some epitopes in common between them. Hence, polyclonal antibody directed against the E. coli γ/τ subunits was raised in rabbits for use in probing T.th. cells by Western analysis. Fig. 7 shows the results of a Western analysis of whole T.th. cells lysed in SDS. The results show that in T.th. cells, the antibody is rather specific for two high molecular proteins which migrate in the vicinity of the molecular masses of E. coli γ and τ subunits.

EXAMPLE 8 Homolog of T. th. γ/τ to dnaX gene products of other organism The Xbal insert encoded an open reading frame, starting with a GTG codon, of 529 amino acids in length (58.0 kDa), closer to the predicted length of the B. subtilis τ subunit (563 amino acids, 62.7 kDa mass)(Alonso et. al., 1986) than the E. coli τ subunit (71.1 kDa)(Yin et. al., 1986). dnaX encoding the γ/τ subunits of E. coli DNA polymerase III holoenzyme is homologous to the holB gene encoding the δ' subunit of the γ complex clamp loader, and this homology extends to all 5 subunits of the eukaryotic RFC clamp loader as well as the bacteriophage gene protein 44 of the gp44/62 clamp loading complex (O'Donnell et. al., 1993). These gene products show greatest homology over the N-terminal 166 amino acid residues (of E. coli dnaX); the C-terminal regions are more divergent. Fig. 4 shows an alignment of the amino acid sequence of the N-terminal regions of the Tth dnaX gene product to those of several other bacteria. The consensus GXXGXGKT motif for nucleotide binding, is conserved in all these protein products. Further, the E. coli δ' crystal structure reveals one atom of zinc coordinated to four Cys residues (Guenther, 1996). These four Cys residues are conserved in the E. coli dnaX gene, and the γ and τ subunits encoded by E. coli dnaX bind one atom of zinc (J. Turner and M. O'Donnell, unpublished). These Cys residues are also conserved in Tth dnaX (shown in Fig. 4). Overall, the level of amino acid identity relative to E. coli dnaX in the N-terminal 165 residues of Tth dnaX is 53 %. The Tth dnaX gene is just as homologous to the B. subtilis dnaX (53 % identity) gene relative to E. coli dnaX. After this region of homology. the C-terminal region of Tth dnaX shares 26% and 20% identity to E. coli and B. subtilis dnaX, respectively. A proline rich region, downstream of the conserved region, is also present in Tth dnaX (residues 346-375), but not in the B. subtilis dnaX (see Figs. 3A and 3B). The overall identity between E. coli dnaX and Tth dnaX over the entire gene is 34%. Identity of Tth. dnaX to B. subtilis dnaX over the entire gene is 28%.

Comparison of dnaX genes from T.th. and E. coli The above identifies a homologue of the dnaX gene of E. coli in Thermus thermophilus. Like the E. coli gene, Tth. dnaX encodes two related proteins through use of a highly efficient translational frameshift. The T.th. γ/τ subunits are tetramers, or mixed tetramers. similar to the γ and τ subunits of E. coli. Further, the γ/τ subunit is a DNA stimulated ATPase like its E. coli counterpart. As expected for proteins from a thermophile. the T.th. γ/τ ATPase activity is thermostabile and resistant to added salt. In E. coli, γ is a component of the clamp loader, and the τ subunit serves the function of holding the clamp loading apparatus together with two DNA polymerases for coordinated replication of duplex DNA. The presence of γ in T.th. suggests it has a clamp loading apparatus and thus a clamp as well. The presence of the τ subunit T.th. implies that T.th. contains a replicative polymerase with a structure similar to that of E. coli DNA polymerase III holoenzyme.

A significant difference between E. coli and T.th. dnaX genes is in the translational frameshift sequence. In E. coli, the heptamer frameshift site contains six A residues followed by a G residue in the context A AAA AAG. This sequence satisfies the X XXY YYZ rule for - 1 frameshifting. The frameshift is made more efficient by the absence of the AAG tRNA for Lys which presumably leads to stalling of the ribosome at the frameshift site and increases the efficiency of frameshifting (Tsuchihashi and Brown, 1992). Two additional aids to frameshifting include a downstream haiφin, and an upstream Shine-Dalgarno sequence (Tsuchihashi and Kornberg, 1990; Larsen et. al., 1994). The -1 frameshift leads to incoφoration of one unique residue at the C-terminus of E. coli γ before encounter with a stop codon.

In T.th.. the dnaX frameshifting heptamer is A AAA AAA. and it is flanked by two other A residues, one on each side. There is also a downstream region of secondary structure. The nearest downstream stop codon is positioned such that gamma would contain only one unique amino acid, as in E. coli. However, the T.th. stop codon is in the -2 reading frame thus requires a -2 frameshift. No precedent exists in nature for -2 frameshifting, although -2 frameshifting has been shown to occur in test cases (Weiss et. al., 1987). In vivo analysis of the T.th. frameshift sequence shows that this natural sequence promotes both -1 and -2 frameshifting in E. coli. Whereas the -2 frameshift results in only one unique C-terminal residue, a -1 frameshift would result in an extension of 12 C-terminal residues. At present, the results do not discriminate which path occurs in T.th.. a -1 or -2 frameshift, or a combination of the two. There are two Shine-Dalgarno sequences just upstream of the frameshift site in T.th. dnaX. In two cases of frameshifting in E. coli, an upstream Shine-Dalgarno sequence has been shown to stimulate frameshifting (reviewed in Weiss et. al., 1897). In release factor 2 (RF2), the Shine-Dalgarno is 3 nucleotides upstream of the shift site, and it stimulates a +1 frameshift event. In the case of E. coli dnaX, a Shine-Dalgarno sequence 10 nucleotides upstream of the shift sequence stimulates the -1 frameshift. One of the T.th. dnaX Shine-Dalgarno sequences is immediately adjacent to the frameshift sequence with no extra space, the other is 22 residues upstream of the frameshift site. Which of these Shine-Dalgarno sequences plays a role in T.th. dnaX frameshifting, if any. will require future study.

In E. coli, efficient separation of the two polypeptides, γ and τ, is achieved by mutation of the frameshift site such that only one polypeptide is produced from the gene (Tsuchihashi and Kornberg, 1990). Substitution of G-to-A in two positions of the heptamer of T.th. dnaX eliminates frameshifting and thus should be a source to obtain τ subunit free of γ. To produce pure γ subunit free of τ, the frameshifting site and sequence immediately downstream of it can be substituted for an in frame sequence with a stop codon.

Examination of the B. subtilis dnaX gene shows no frameshift sequence that satisfies the X XXY YYZ rule. Hence, it would appear that dnaX does not make two proteins in this gram positive organism.

Rapid thermal motions associated with high temperature may make coordination of complicated processes more difficult. It seems possible that organizing the components of the replication apparatus may become yet more important at higher temperature. Hence, production of a τ subunit that could be used to crosslink two polymerases and a clamp loader into one organized particle may be most useful at elevated temperature. As stated above, the following examples describe the continued isolation and purification of the substantial entirety of the Polymerase III from the extreme thermophile Thermus thermophilus. It is to be understood that the following exposition is reflective of the protocol and characteristics, both moφhological and functional, of the Polymerase Ill-type enzymes that are the focus of the present invention, and that the invention is hereby illustrated and comprehends the entire class of enzymes of thermophilic origin.

EXAMPLE 9

Purification of the Thermus thermophilus DNA polymerase III All steps in the purification assay were performed at 4°C. The following assay was used in the purification of DNA polymerase from T th. cell extracts. Assays contained 2.5 mg activated calf thymus DNA (Sigma Chemical Company) in a final volume of 25 ml of 20 mM Tris-Cl (pH 7.5), 8 mM MgCl₂, 5 mM DTT, 0.5 mM EDTA, 40 mg/ml BSA, 4% glycerol, 0.5 mM ATP. 3 mM each dCTP,. dGTP, dATP, and 20 mM [α-³²P]dTTP. An aliquot of the fraction to be assayed was added to the assay mixture on ice followed by incubation at 60°C for 5 min. DNA synthesis was quantitated using DE81 paper followed by washing off unincorporated nucleotide. Incoφorated nucleotide was determined by scintillation counting of the filters.

Thermus thermophilus cell extracts were prepared by suspending 35 grams of cell paste in 200 ml of 50 mM TRIS-HCl, pH=7.5, 30 mM spermidine, 100 mM NaCl, 0.5 mM EDTA, 5 mM DTT, 5% glycerol, followed by disruption by passage through a French pressure cell (15,000 PSI). Cell debris was removed by centrifugation (12.000 RPM. 60 min). DNA polymerase III in the clarified supernatant was precipitated by treatment with ammonium sulphate (0.226 gm/liter) and recovered by centrifugation. This fraction was then backwashed with the same buffer (but lacking spermidine) containing 0.20 gm 1 ammonium sulfate. The pellet was then resuspended in buffer A and dialyzed overnight against 2 liters of buffer A; a precipitate which formed during dialysis was removed by centrifugation (17,000 RPM. 20 min).

The clarified dialysis supernatant, containing approximately 336 mg of protein, was applied onto a 60 ml heparin agarose column equilibrated in buffer A which was washed with the same buffer until A280 reached baseline. The column was developed with a 500 ml linear gradient of buffer A from 0 to 500 mM NaCl. More tightly adhered proteins were washed off the column by treatment with buffer A (20 mM Tris Hcl, pH = 7.5, 0.1 mM EDTA, 5mM DTT, and 10% glycerol) and 1M NaCl. Some DNA polymerase activity flowed through the column. Two peaks (HEP. PI and HEP.P2) of DNA polymerase activity eluted from the heparin agarose column containing 20 mg and 2 mg of total protein respectively (Fig. 13 A). These were kept separate throughout the remainder of the purification protocol.

The Pol III resided in HEP.P 1 as indicated by the following criteria: 1 ) Western analysis using antibody directed against the α subunit of E. •coli Pol III indicated presence of Pol III in HEP.P 1, 2) Only the HEP.P 1 fraction was capable of extending a single primer around an M13mpl 8 7.2 kb ssDNA circle (explained later in Example 14). This type of long primer extension is a characteristic of Pol III type enzymes. 3) Only the HEP.P 1 provided DNA polymerase activity that was retained on an ATP-agarose affinity column. This is indicative of a Pol Ill-type DNA polymerase since the γ and τ subunits are ATP interactive proteins.

The first peak of the heparin agarose column (HEP. PI : 20 mg in 127.5 ml) was dialyzed against buffer A and applied onto a 2ml N6-linkage ATP agarose column pre-equilibrated in the same buffer. Bound protein was eluted by a slow (0.05 ml/min) wash with buffer A + 2M NaCl and collected into 200 μl fractions.

Chromatography of peak HEP.P 1 yielded a flow-through (HEP.P 1-ATP-FT) and a bound fraction (HEP.P 1-ATP-Bound) (Fig. 13B). Binding of peak HEP.P2 to the ATP column could not be detected, though DNA polymerase activity was recovered in the flow-through.

The HEP.P 1-ATP-Bound fractions from the ATP agarose chromatographic step were further purified by anion exchange over monoQ. The HEP.P 1-ATP-Bound fractions were diluted with buffer A to approximately the conductivity of buffer A plus 25 mM NaCl and applied to a 1ml monoQ column equilibrated in Buffer A. DNA polymerase activity eluted in the flow-through and in two resolved chromatographic peaks (MONOQ peakl and peak2) (Fig. 13C). Peak 2 was by far the major source of DNA polymerase activity. Western analysis using rabbit antibody directed against the E. coli a subunit confirmed presence of the subunit in the second peak (see the

Western analysis in Fig. 14B). Antibody against the E. coli γ subunit also confirmed the presence of the γ subunit in the second peak (not shown). Some reaction against and γ was also present in the minor peak (first peak). The Coomassie Blue SDS polyacrylamide gel of the MonoQ fractions (Fig. 14A) showed a band that co- migrated with E. coli a and was in the same postion as the antibody reactive material (antibody against E. coli a). Also present are bands corresponding to τ, , δ and δ'. These subunits. along with ^β. are all that is necessary for rapid and processive synthesis and primer extension over a long (> 7 kb) stretch of ssDNA in the case ofE. coli DNA Polymerase III holoenzyme.

The Pol Ill-type enzyme purified from T.th. may be a Pol III*-like enzyme that contains the DNA polymerase and clamp loader subuits (i.e. like the Pol III* of E. coli). The evidence for this is: 1) the presence of dnaX and dnαE gene products in the same column fractions as indicated by Western analysis (see above); 2) the ability of this enzyme to extend a primer around a 7.2 kb circular ssDNA upon adding only β (see Example 14); 3) stimulation of Pol III by adding β on linear DNA, indicating β subunit is not present in saturating amounts (see Example 13); and 4) the presence of τ in T.th. which may glue the polymerase and clamp loader into a Pol III* as in E. coli; and 5) the comigration of α with subunits γ, δ and 6" of the clamp loader in the column fractions of the last chromatographic step (MonoQ. Fig. 14A)..

Micro-sequencing of T. th DNA Polymerase III α subunit The subunit from the purified T.th DNA polymerase III (HEP.P 1.ATP-Bound.MONOQ peak2) was blotted onto PVDF membrane and was cut out of the SDS-PAGE gel and submitted to the Protein-Nucleic Acid Facility at Rockefeller University for N-terminal sequencing and proteolytic digestion, purification and microsequencing of the resultant peptides. Analysis of the a candidate band (Mw = 130kD) yielded four peptides, two of which (TTH1, TTH2) showed sequence similarity to α subunits from various bacterial sources (see Fig. 15).

EXAMPLE 10

Identification of the Thermits thermophilus dnaE gene encoding the subunit of DNA polymerase III holoenzyme

Cloning of the dnaE gene was started with the sequence of the TTH1 peptide from the purified subunit (FFIEIQNHGLSEQK). The fragment was aligned to a region at approximately 180 amino acids downstream of the N-termini of several other known subunits as shown in Fig. 15. The upstream 33mer

(5'-GTGGGATCCGTGGTTCTGGATCTCGATrτAAOAA-3') consists of a BamHI site within the first 9 nucleotides (underlined) and the sequence coding for the following peptide HGLSEQK on the complementary strand. The downstream 29mer

(5'-GTGGGATCCACGGSCTSTCSGAGCAGAAG-3') consists of a BamHI site within the first 9 nucleotides (underlined) and the following sequence coding for the peptide FFIEIQNH.

These two primers were directed away from each other for the puφose of perfoming inverse PCR (also called circular PCR). The amplification reactions contained lOng

T.th. genomic DNA (that had been cut and religated with Xmal), 0.5 mM of each primer, in a volume of 100 μl of Vent polymerase reaction mixture containing 10 μl ThermoPol Buffer. 0.5 mM of each dNTP and 0.25 mM MgS0₄. Amplification was performed using the following cycling scheme:

1. 4 cycles of: 95.5°C - 30". 45°C - 30", 75°C - 8'

2. 6 cycles of: 95.5°C - 30", 50°C - 30". 75 °C - 6' 3. 30 cycles of: 95.5 °C - 30". 52.5°C - 30", 75°C - 5'

A 1.4kb fragment was obtained and cloned into pBS-SK:BamHI (i.e. pBS-SK (Stratragene) was cut with BamHI). This sequence was bracketted by the 29mer primer on both sides and contained the sequence coding for the N-terminal part of the a subunit up to the peptide used for primer design.

To obtain further dnaE gene sequence, the 7TH2 peptide was used. It was aligned to a region about 600 amino acids from the N-termini of the other known α subunits (Fig. 15B).

The upstream 34mer (5'-GCGGGATCCTCAACGAGGACCTCTCCATCTTCAA-3' consists of a BamHI site within the first 9 nucleotides (underlined) and the sequence from the end of the fragment previously obtained. The downstream 33mer

(5'-GCGGGATCCTTGTCGTCSAGSGTSAGSGCGTCGTA-3') consists of a BamHI site within the first 9 nucleotides (underlined) and the following sequence coding for the peptide YDALTLDD on the complementary strand. The amplification reactions contained 10 ng T.th. genomic DNA, 0.5 mM of each primer, in a volume of 100 μl of Vent polymerase reaction mixture containing 10 μl ThermoPol Buffer, 0.5 mM of each dNTP and 0.25 mM Mg S0₄. Amplification was performed using the following cycling scheme:

1. 4 cycles of: 95.5°C - 30", 45°C - 30", 75°C - 8'

2. 6 cycles of: 95.5°C - 30", 50°C - 30", 75°C - 6' 3. 30 cvcles of: 95.5°C - 30". 55 °C - 30". 75°C - 5' A 1.2kb PCR fragment was obtained and cloned into pUC19:BamHI. The fragment was bracketted by the downstream primer on both sides and contained the region overlapping in 56 bp with the fragment previously cloned.

To obtain yet more dnaE sequence, the following primers were used. The upstream 39mer (3^'-GTGTGGATCCTCGTCCCCCTCATGCGCGACCAGGAAGGG-5^ consists of a BamHI site within the first 10 nucleotides (underlined) and the sequence from the end of the fragment previously obtained. The downstream 27mer (5'-GTGTGGATCCTTCTTCTTSCCCATSGC-3') consists of a BamHI site within the first 10 nucleotides (underlined), and the sequence coding for the peptide AMGKKK (at position approximately 800 residues from the N terminus) on the complementary strand. The AMGKKK sequence was chosen for primer design as it is highly conserved among the known gram-negative α subunits. The amplification reactions contained 10 ng T.th. genomic DNA, 0.5 mM of each primer, in a volume of 100 μl of Taq polymerase reaction mixture containing 10 μl PCR Buffer. 0.5 mM of each dNTP and 2.5 mM MgCl₂. Amplification was performed using the following cycling scheme:

1. 3 cycles of: 95.5°C - 30", 45°C - 30". 72°C - 8'

2. 6 cycles of: 94.5°C - 30". 55 °C - 30", 72°C - 6'

3. 32 cycles of: 94.5°C - 30", 50°C - 30", 72°C - 5'

A 2.3kb PCR fragment was obtained instead of the expected 0.6 kb fragment. BamHI digestion of the PCR product resulted in three fragments of 1.1 kb, 0.7kb and 0.5kb. The 1.1 kb fragment was cloned into pUC19:BamHI. It turned out to be the one adjacent to the fragment previously obtained and contained the dnaE sequence right up to the region coding for the AMGKKK peptide. but was disrupted by an intein just upstream of this region.

The sequence that follows this was amplified from the 2.3kb original PCR product using the same conditions and cycling scheme as for the 2.3kb fragment. The downstream primer was the same as in the previous step. The upstream 27mer (3'-GTGTGGATCCGTGGTGACCTTAGCCAC-5- consisted of a BamHI site within the first 9 nucleotides (underlined) and the sequence from the end of the l .lkb fragment previously described.

The expected 1.2kb PCR fragment was obtained and cloned into pUC 19:SmaI. This fragment coded for the rest of the intein and the end of it was used to obtain the next sequence of dnaE downstream of this region. The upstream 30mer (3'-TTCGTGTCCGAGGACCTTGTGGTCCACAAC-5^•) was a sequence from the end of the intein. The downstream 23mer (5'-CCAGAATCGTCTGCTGGTCGTAG-3') was the sequence from the end of the dnaE gene of D.rad. (coding on the complementary strand for the region slightly homologous in the distantly related α subunits and possibly highly homologous between T.th. and D.rad. a subunits). The amplification reactions contained 10 ng T.th. genomic DNA. 0.5 mM of each primer, in a volume of 100 μl of Vent polymerase reaction mixture containing 10 μl ThermoPol Buffer, 0.5 mM of each dNTP and 0.1 mM Mg S0₄. Amplification was performed using the following cycling scheme:

1. 3 cycles of: 95.5°C - 30". 55 °C - 30". 75°C - 8'

2. 32 cycles of: 94.5°C - 30". 50°C - 30". 75 °C - 5'

A 2.5kb PCR fragment was obtained and cloned into pUC19:SmaI. This fragment contained the dnaE sequence coding for the 300 mino acids next to the AMGKKK region disrupted by yet a second intein inside another sequence that is conserved among the known α subunits (FNKSHSAAY).

To obtain the rest of the dnaE gene the upstream 19mer (5'-AGCACCCTGGAGGAGCTTC-3') from the end of the known dnaE sequence was used. The downstream primer was: 5'-CATGTCGTACTGGGTGTAC-3'. The amplification reactions contained 10 ng T.th. genomic DNA, 0.5 mM of each primer, in a volume of 100 μl of Vent polymerase reaction mixture containing 10 μl ThermoPol Buffer. 0.5 mM of each dNTP and 0.1 mM Mg S0₄. Amplification was performed using the following cycling scheme:

1. 3 cycles of: 95.5°C - 30". 55°C - 30". 75°C - 8' 2. 32 cycles of: 94.5°C - 30". 50°C - 30", 75°C - 5'

A l .Okb fragment bracketed by this upstream primer was obtained. It contained the 3' end of the dnaE gene.

EXAMPLE 11

Cloning and Expression of the Thermus thermophilus dnaQ gene encoding the e subunit of DNA polvmerase III holoenzyme

Cloning of dnaQ - The DnaQ gene of E. coli and the corresponding region of PolC of B. subtilis, evolutionary divergent organisms, share approximately 30% identity. Comparison of the predicted amino acid sequences encoded by DnaQ of E. coli and PolC of B. subtilis revealed two highly conserved regions positions (Fig. 17). Within each of these regions, a nine amino acid sequence was used to design two oligonucleotide primers for use in the polymerase chain reaction.

The regions highly conservative among Pol III exonucleases were chosen to design the degenerate primers for the amplification of a T.th. dnaQ internal fragment (see Fig. 17). DNA oligonucleotides for amplification of T.th. genomic DNA were as follows. The upstream 27mer (5'-GTSGTSNNSGACNNSGAGACSACSGGG-3') encodes the following sequence (VVXDXETTG). The downstream 27mer (5'-GAASCCSNNGTCGAASNNGGCGTTGTG-3') encodes the sequence HNAXFDXGF on the complementary strand. The amplification reactions contained 10 ng T.th. genomic DNA, 0.5 mM of each primer, in a volume of 100 μl of Vent polymerase reaction mixture containing 10 μl ThermoPol Buffer, 0.5 mM of each dNTP and 0.5 mM MgS0 . Amplification was performed using the following cycling scheme:

1. 5 cycles of: 95.5°C - 30", 40°C - 30". 72°C - 2'

2. 5 cycles of: 95.5°C - 30", 45°C - 30", 72°C - 2' 3. 30 cycles of: 95.5°C - 30", 50°C - 30", 72°C - 30"

Products were visualized in a 1.5 % native agarose gel. A fragment of the expected size of 270 bp was cloned into the Smal site of pUC 19 and sequenced with the CircumVent Thermal Cycle DNA sequencing kit accordinig to the manufacturer's instructions (New England Biolabs).

To obtain further sequence of the dnaQ gene, genomic DNA was digested with either mhol. BamHI, Kpnl or Ncol. These restriction enzymes were chosen because the cut T.th. genomic DNA frequently. 0.1 μg of DNA for each digest was ligated by T4 DNA ligase in 50 μl of ligation buffer (50 mM Tris-HCl (pH 7.8), 10 mM MgCl₂, 10 mM dithiothreitol. 1 mM ATP, 25 mg/ml bovine serum albumin) overnight at 20 °C. The ligation mixtures were used for cicular PCR.

DNA oligonucleotides for amplification of T.th. genomic DNA were the following. The upstream 27mer (5'-CGGGGATCCACCTCAATCACCTCGTGG-3') consists of a BamHI site within the first 9 nucleotides (underlined) and the sequence complementary to 42-6 lbp region of the previously cloned dnaQ fragment. The downstream 30mer (5^,-CGGGjGATCCGCCACCTTGCGGCTCCGGGTG-3') consists of a BamHI site within the first 9 nucleotides (underlined) and the sequence corresponding to 240-261 bp region of the dnaQ fragment (see Fig. 17).

The amplification reactions contained 1 ng T.th. genomic DNA (that had been cut with Ncol and religated into circular DNA for circular PCR), 0.4 mM of each primer, in a volume of 100 μl of Vent polymerase reaction mixture containing 10 μl ThermoPol Buffer. 0.5 mM of each dNTP. 0.5 mM MgS0₄. and 10% DMSO. Circular amplification was performed using the following cycling scheme:

1. 5 cycles of: 95.5°C - 30". 50°C - 30", 72°C - 8'

2. 35 cycles of: 95.5°C - 30", 55°C - 30", 72°C - 6' 3. 72°C - 10'

A 1.5 kb fragment was obtained and cloned into the BamHI site of the pUC19 vector. Partial sequencing of the fragment reveiled that it contained the dnaQ regions adjacent to sequences corresponding to the PCR primers and hence contained the sequences both upstream and downstream of the previously cloned dnaQ fragment. One of Ncol sites turned out to be approximatly 300 bp downstream of the end of the first cloned dnaQ sequence and hence did not include the 3' end of dnaQ. To obtain the 3' end. another inverse PCR reaction was performed. Since an Apal restiction site was recognized within this newly sequenced dnaQ fragment, the circular PCR procedure was performed using as template an Apal digest of T.th. genomic DNA that was ligated (circularized) under the same conditions as described above.

DNA oligonucleotides for amplification of the Apal/religated T.th. genomic DNA were as follows. The upstream 3 lmer

(5'-GCGCJT TAGACGAGTTCCCAAAGCGTGCGGT-3') consists of a mbal site within the first 10 nucleotides (underlined) and the sequence complementary to the region downstream of the Apal restriction site in the newly sequenced dnaQ fragment. The downstream 3 lmer f5'-CGCGTCTAGATCACCTGTATCCAGA-3') consists of a Xbal site within the first 10 nucleotides (underlined) and the sequence corresponding to another region downstream of the Apal restriction site in the newly sequenced dnaQ fragment. The 1.7 kb PCR fragment was cloned into the Xbal site of the pUC19 vector and partially sequenced. The sequence of dnaQ, and the protein sequence of the e subunit encoded by it. is shown in Fig. 18. The dnaQ gene is encoded by an open reading frame of 209 (or 190 depending on which Val is used as the initiating residue) amino acids in length (23598.5 kDa - or 21383.8 kDa for shorter version), similar to the length of the E.coli e subunit (243 amino acids, 27099.1 kDa mass) (see Fig. 17).

The entire amino acid sequence of the e subunit predicted from the T.th. dnaQ gene aligns with the predicted amino acid sequence of the dnaQ genes of other organisms with only a few gaps and insertions (the first two amino acids, and four positions downstream) (Fig. 17). The consensus motifs (VVXDXETTG, HNAXFDXGF. and HRALYD), characteristic for exonucleases, are conserved. Overall, the level of amino acid identity relative to most of the known e subunits, or corresponding proofreading exonuclease domains of gram positive PolC genes is approximately 30%. Upstream of start 1 (Fig. 17) there were stop codons in all three reading frames.

Expression of DnaQ - The DnaQ gene was cloned gene into the pET24-a expression vector in two steps. First, the PCR fragment encoding the N-terminal part of the gene was cloned into the pUC19 plasmid, containing the Apal inverse PCR fragment into Ndel/Apal sites. DNA oligonucleotides for amplification of T.th. genomic DNA were as follows. The upstream 33mer

(5'-GCGGCGCArA GGTGGTGGTCCTGGACCTGGAG-3') consists of an Ndel site within the first 12 nucleotides (underlined) and the begining of the dnaQ gene. The downstream 31 mer (5'-CGCGTCJ_AGATCACCTGTATCCAGA-3'), already used for Apal circular PCR, consists of an Xbal site within the first 10 nucleotides (underlined) and the sequence corresponding to the region downstream of the Apal restriction site. The 2.2 kb Ndel/Sall fragment was then cloned into the Ndel/Xhol sites of the pET16 vector to produce pET2A-a:dnaQ . The e subunit was expressed in the BL21 LysS strain transformed by the pE12A-a:dnαQ plasmid.

EXAMPLE 12 The Thermits thermophilus dnaN gene encoding the β subunit of DNA polymerase III holoenzvme

Strategy of cloning DnaN by use of DnaA - DnaN proteins are highly divergent in bacteria making it difficult to clone them by homology. The level of identity between DnaN representatives from E. coli and B. subtilis is as low as 18%. These 18% of identical amino acid residues are dispersed through the proteins rather then clustering together in conservative regions, further complicating use of homology to design PCR primers. However, one feature of dnaN genes among widely different bacteria is their location in the chromosome. They appear to be near the origin, and immediately adjacent to the dnaA gene. DnaA genes show good homology among different bacteria and thus we first cloned dnaA in order to obtain a DNA probe that is likely near dnaN.

Identification of dnaA and dnaN - The DnaA genes of E. coli and B. subtilis share 58% identity at the amino acid sequence level within the ATP -binding domain (or among the representatives of gram-positive and gram-negative bacteria, evolutionary divergent organisms). Comparison of the predicted amino acid sequences encoded by dnaA of E. coli and B. subtilis revealed two highly conserved regions (Fig. 19). Within each of these regions, a seven amino acid sequence was used to design two oligonucleotide primers for use in the polymerase chain reaction. The DNA oligonucleotides for amplification of T.th. genomic DNA were as follows. The upstream 20mer (5'-GTSCTSGTSAAGACSCACTT-3') encodes the following sequence: VLVKTHL. The downstream 2 lmer

(5'-SAGSAGSGCGTTGAASGTGTG-3') encodes the sequence: HTFNALL, on the complementary strand. The amplification reactions contained 10 ng T.th. genomic DNA. 0.5 mM of each primer, in a volume of 100 μl of Vent polymerase reaction mixture containing 10 μl ThermoPol Buffer, 0.5 mM of each dNTP and 0.5 mM MgS0₄. Amplification was performed using the following cycling scheme: 1. 5 cycles of: 95.5°C - 30", 45 °C - 30", 75°C - 2' 2. 5 cycles of: 95.5 °C - 30". 50°C - 30". 75 °C - 2'

3. 30 cycles of: 95.5°C - 30", 52°C - 30", 75 °C - 30"

Products were visualized in a 1.5% native agarose gel. A fragment of the expected size of 300 bp was cloned into the Smal site of pUC19 and sequenced with the CircumVent Thermal Cycle DNA sequencing kit (New England Biolabs).

To obtain a larger section of the T.th. dnaA gene, genomic DNA was digested with either Haell, Hindlll. KasI, Kpnl, Mlul. Ncol, NgoMI, Nhel, Nsil, PaeR7I, PstI, Sad. Sail, Spel, SphI, Stul. or Xhol, followed by Southern analysis in a native agarose gel. The filter was probed with the 300 bp PCR product radiolabeled by random priming. Four different restriction digests showed a single fragment of reasonable size for further cloning. These were. KasI, NgoMI, and Stul which produced fragments of about 3 kb. and Ncol that produced a 2kb fragment. Also, a Kpnl digest resulted in two fragments of about 1.5 kb and 10 kb.

Genomic DNA digests using either NgoMI and Stul were used to obtain the dnaA gene by inverse PCR (also referred to as circular PCR). In this procedure, 0.1 μg of DNA from each digest was treated separately with T4 DNA ligase in 50 μl of ligation buffer (50 mM Tris-HCl (pH 7.8), 10 mM MgCL. 10 mM dithiothreitol. 1 mM ATP. 25 mg/ml bovine serum albumin) overnight at 20 °C. This results in circularizing the genomic DNA fragments. The ligation mixtures were used as substrate in inverse PCR.

DNA oligonucleotides for amplification of recircularized T.th. genomic DNA were as follows. The upstream 22mer was 5'-CTCGTTGGTGAAAGTTTCCGTG-3'. and the downstream 24mer was 5'-CGTCCAGTTCATCGCCGGAAAGGA-3'. The amplification reactions contained 5 ng T.th. genomic DNA. 0.5 μM of each primer, in a volume of 100 μl of Taq polymerase reaction mixture containing 10 μl PCR Buffer. 0.5 mM of each dNTP and 2.5 mM MgCl_:. Amplification was performed using the following cycling scheme:

1. 5 cycles of: 95.0°C - 30". 55°C - 30", 72°C - 10'

2. 35 cycles of: 95.5°C - 30". 50°C - 30", 72°C - 8'

The PCR fragments of the expected length for NgoMI and Stul treated and then ligated chromosomal DNA were digested with either BamHI or Sau3a and cloned into pUC19 : BamHI and pUC19:(BamHI+SmaI) and sequenced with CircumVent Thermal Cycle DNA sequencing kit. The 1.6kb (BamHI+BamH) fragment from the NgoMI PCR product contained a sequence coding for the N-terminal part of DnaN, followed by the gene for enolase. The lkb (Sau3a+Sau3a) fragment from the same PCR product included the start of dnaN gene and sequence characteristic of the origin of replication (i.e. 9mer DnaA-binding site sequences). The 0.6kb (BamHI+BamHI) fragment from the Stul PCR reaction contained starts for dnaA and gidA genes in inverse orientation to each other. The 0.4 kb (Sau3a+Sau3a) fragment from the same PCR product contained the 3' end of the dnaA gene and DNA sequence characteristic for the origin of replication.

This sequence information provided the beginning and end of both the dnaA and the dnaN genes. Hence, these genes were easily cloned from this information. Further, the DnaN gene was readily cloned and expressed in a pET24-a vector. These steps are described below.

Cloning and sequence of the dnaA gene - The dnaA gene was cloned for sequencing in two parts: from the potential start of the gene up to its middle and from the middle up to the end. For the N-terminal part the upstream 27mer (5'-TCTGGCAACACGTTCTGGAGCACATCC-3') was 20 bp downsteam of the potential start codon of the gene. The downstream 23 mer

(5'-TGCTGGCGTTCATCTTCAGGATG-3') was approximately from the middle of the dnaA gene. For the C-terminal part the upstream 23mer (5'-CATCCTGAAGATGAACGCCAGCA-3') was complementary to the previous primer. The downstream 25 mer (5'-AGGTTATCCACAGGGGTCATGTGCA-3') was 20 bp upstream the potential stop codon for the dnaA gene. The amplification reactions contained 10 ng T.th. genomic DNA. 0.5 μM of each primer, in a volume of 100 μl of Vent polymerase reaction mixture containing 10 μl ThermoPol Buffer, 0.5 mM of each dNTP and 0.5 mM MgS0₄. Amplification was performed using the following cycling scheme:

1. 5 cycles of: 95.5°C - 30", 55°C - 30", 75°C - 3' 2. 30 cycles of: 95.5°C - 30", 50°C - 30", 75 °C - 2'

Products were visualized in a 1.0% native agarose gel. Fragments of the expected sizes of 750 bp and 650 bp were produced, and were sequenced using CircumVent Thermal Cycle DNA sequencing method (New England Biolabs). The nucleotide and amino acid sequences of dnaA and its protein product are shown in Fig. 20. The DnaA protein is homologous to the DnaA proteins of several other bacteria as shown in Fig. 19.

Cloning and expression of dnaN - The full length dnaN gene was obtained by PCR from T.th. total DNA. DNA oligonucleotides for amplification of T.th. dnaN were the following: the upstream 29mer (5'-GTGTGTCATATGAACATAACGGTTCCCAA-3') consists of an Ndel site within first 1 1 nucleotides (underlined), followed by the sequence for the start of the dnaN gene; the downstream 29mer

(5'-GCGCO ATTCTCCCTTGTGGAAGGCTTAG-3') consists of an EcoRI site within the first 10 nucleotides (underlined), followed by the sequence complementary to a section just downstream of the dnaN stop codon. The amplification reactions contained 10 ng T.th. genomic DNA, 0.5 μM of each primer, in a volume of 100 μl of Vent polymerase reaction mixture containing 10 μl Thermopol Buffer. 0.5 mM of each dNTP and 0.2 mM Mg S0₄. Amplification was performed using the following cycling scheme: 1. 5 cycles of: 95.0°C - 30". 55°C - 30", 75°C - 5^". 2. 35 cycles of: 95.5 °C - 30". 50°C - 30", 75 °C - 4'.

The nucleotide and amino acid sequences of dnaN and the β subunit. respectively, are shown in Fig. 21. The T.th. β subunit shows limited homology to the β subunit sequences of several other bacteria over its entire length (Fig. 22).

The approximately 1 kb dnaN gene was cloned into the pET24-a expression vector using the Ndel and EcoRI restriction sites both in the dnaN containing PCR product and in pEt24-a (Fig. 23). Expression of T.th. β subunit was obtained under the following conditions: a fresh colony of B121(DE3) E.coli strain was transformed by the pET24-a:dnaN plasmid, and then was grown in LB broth containing 50 mg/ml kanamycin at 37°C until the cell density reached 0.4 OD₆₀₀ The cell culture was then induced for dnaN expression upon addition of 2 mM IPTG. Cells were harvested after 4 additional hours of growth under 37°C. The induction of the T.th. β subunit is shown in Fig. 24.

Two liters of BL21 (DE3)pET<i«αNcells were grown in LB media containing 50 mg/ml ampicillin at 37°C to an O.D. of 0.8 and then IPTG was added to a concentration of 2 mM. After a further 2 h at 37°C. cells were harvested by centrifugation and stored at -70°C. The following steps were performed at 4°C. Cells were thawed and resuspended in 40 ml of 5 mM Tris-HCl (pH 8.0), 1% sucrose. IM ΝaCl, 5 mM DTT. and 30 mM spermidine. Cells were lysed using a French

Pressure cell at 20.000 psi. The lysate was allowed to sit at 4°C for 30 min. and then cell debris was removed by centrifugation (Sorvall SS-34 rotor, 45 min. 18,000 φm). The supernatant was incubated at 65 °C for 20 minutes with occasional stirring. The resulting protein precipitate was removed by centrifugation as described above. The supernatant was dialyzed against 4 liters of buffer A containing 50 mM ΝaCl overnight. The dialyzed supernatant was clarified by centrifugation (35 ml, 150 mg total) and then loaded onto an 8 ml MonoQ column equilibrated in buffer A containing 50 mM NaCl. The column was washed with 5 column volumes of the same buffer and then eluted with a 120 ml gradient of buffer A plus 50 mM NaCl to buffer A plus 500 mM NaCl. Fractions of 2 ml were collected. Over 50 mg of T.th. β was recovered in fractions 5-21.

EXAMPLE 13

Alternate synthetic path in absence of clamp loader activity.

As discussed earlier, the Pol Ill-type enzyme of the present invention is capable of application and use in a variety of contexts, including a method wherein the clamp loader component that is traditionally involved in the initiation of enzyme activity, is not required. The clamp loader generally functions to increase the efficiency of ring assembly onto circular primed DNA because both the ring and the DNA are circles and one must be broken transiently for them to become interlocked rings. In such a reaction, the clamp loader increases the efficiency of opening the ring.

The procedure described below illustrates the instance where the clamp loader need not be present. For example, the β clamp can be assembled onto DNA in the absence of the clamp loader. Particularly, the bulk of primed templates in PCR reactions are linear ssDNA fragments that are primed at the ends. On linear primed DNA. the ring need not open at all. Instead, the ring can simply thread onto the end of the linear primed template (Bauer and Burgers, 1988; Tan et. al, 1986; O'Day et. al., 1992; Burgers and Yoder. 1993). Hence, on linear primed templates, such as those generated in PCR. the beta clamp can simply slide over the DNA end. After the ring slides onto the end. the DNA polymerase can associate with the ring for enhanced DNA synthesis.

Such "end assembly" is common among Pol Ill-type enzymes and has been demonstrated in the yeast and human systems. Rings assembling onto linear DNA for use by their respective DNA polymerases are shown in the following example demonstrated in the E. coli bacterial system, in the human system, and in the T.th. system.

The bulk of the primed templates in PCR reactions are linear ssDNA fragments that are primed at their ends. However, these end primed linear fragments are not generated until after the first step of PCR has already been performed. In the very first step, PCR primers generally anneal at internal sites in a heat denatured ssDNA template. Primed linear templates are then generated in subsequent steps enabling use of this alternate path. For this first step, the clamp may be assembled onto an internal site in the absence of the clamp loader using special conditions that allow clamp assembly in the absence of a clamp loader.

For example, a set of conditions that lead to assembly of the clamp onto circular DNA (i.e. internal primed sites) have been described in the protocol for the use of the bacteriophage T4 ring shaped clamp (gene 45 protein) without the clamp loader (Reddy et. al.. 1993). In this case, polyethylene glycol leads to "macro molecular crowding" such that the clamp and DNA are pushed together in close proximity leading to the ring self assembling onto internal primed sites on circular DNA. Other possible conditions that may lead to assembly of rings onto internal sites include use of a high concentration of beta such that use of heat or denaturant to break the dimeric ring into two half rings (crescents) followed by lowering the heat (or dilution or removal of denaturant) leading to rings assembling around the DNA.

The ring shaped sliding clamps of E. coli and human slide over the end of linear DNA to activate their respective DNA polymerase in the absence of the clamp loader. This clamp loader independent assay is performed in the bacterial system in Fig.25A. For this assay, the linear template is polydA primed with oligodT. The polydA is of average length 4500 nucleotides and was purchased from SuperTecs. 01igodT35 was synthesized by Oligos etc. The template was prepared using 145μl of 5.2 mM (as nucleotide) polydA and 22 μ: of 1.75 mM (as nucleotide) oligodT. The mixture was incubated in a final \ olume of 2100 μ; T.E. buffer (ratio as nucleotide was 21 : 1 polydA to oligodT). The mixture was heated to boiling in a 1 ml Eppendorf tube, then removed and allowed to cool to room temperature. Assays were performed in a final volume of 25 μl 20 mM Tris-Cl (pH 7.5). 8 mM MgCL, 5 mM DTT. 0.5 mM EDTA, 40 mg/ml BSA. 4% glycerol, containing 20 μM [α-³ P]dTTP. 0.1 μg polydA-oligodT. 25 ng Pol III and, where present, 5 ug of β subunit. Proteins were added to the reaction on ice, then shifted to 37 °C for 5 min. DNA synthesis was quantitated using DE81 paper as described (Rowen and Kornberg, 1979).

In the linear template assay, no ATP or dATP is provided and therefore, a clamp loader, even if present, is not active. Thus, the clamp (e.g. β) can only stimulate the DNA polymerase provided the clamp threads onto the DNA (see diagram in Fig. 25). Hence, threading of the clamp is shown by a stimulation of the DNA polymerase. In lane 1 of Fig. 25 A. the DNA polymerase is incubated with the the linear DNA in the absence of the clamp, and lane 2 shows the result of adding, the clamp. The results shov that the clamp is able to thread onto the DNA ends and stimulate the DNA polymerase in the absence of ATP and thus, in the absence of clamp loading as well.

This clamp loader independent assay is performed in the human system in Fig. 25B. The assay reaction (25 μl) contains 50 mM Tris-HCl (pH=7.8), 8 mM MgC12, 1 mM DTT, 1 mM creatine phosphate, 40 μg/ml bovine serum albumin, 0.55 μg human SSB, 100 ng PCNA (where present), 7 units DNA polymerase delta (1 unit incoφorates 1 pmol dTMP in 60 min.), 40 mM [α-'²P]dTTP and 0.1 μg polydA-oligodT. Proteins were added to the reaction on ice, then shifted to 37°C for 60 min. DNA synthesis was quantitated using DES 1 paper as described (Rowen and Kornberg, 1979). In lane 3, (Fig. 25) the DNA polymerase δ is incubated with the linear DNA in the absence of the clamp, and lane 4 showes the result of adding the PCNA clamp. The results demonstrate that the clamp is able to thread onto the DNA ends and stimulate the DNA polymerase in the absence of ATP and thus, the absence of clamp loading.

This clamp loader independent assay is performed in the T.th. system in Fig. 25C. The assay reaction is exactly as described above for use of the E. coli Pol III and beta system except the temperature is 60 °C and here the Pol III is HEP. PI T.th. Pol III (0.5 μl. providing 0.1 units where one unit is equal to 1 pmol of dTTP incoφorated in 1 minute under these conditions and in the absence of beta), and the beta subunit is 7 μg T.th. β (from the MonoQ column). Proteins were added to the reaction on ice, then shifted to 37°C for 60 min. DNA synthesis was quantitated using DE81 paper as described (Rowen and Kornberg, 1979).. In lane 3 (Fig. 25C), the T. Th. Pol III is incubated with the linear DNA in the absence of the clamp, and lane 4 shows the result of adding the T.th. β clamp. The results demonstrate that the clamp is able to thread onto the DNA ends and stimulate the DNA polymerase in the absence of clamp loader activity.

EXAMPLE 14

Use of T.th. Pol III in long chain primer extension

A characteristic of Pol Ill-type enzymes is their ability to extend a single primer for several kilobases around a long (e.g. 7 kb) circular single stranded DNA genome of a bacteriophage. This reaction uses the circular β clamp protein. For the circular β to be assembled onto a circular DNA genome, the circular β must be opened, positioned around the DNA, then closed. This assembly of the circular beta around DNA requires the action of the clamp loader, which uses ATP to open and close the ring around DNA. In this example we use as a template the 7.2 kb circular single strand DNA genome of bacteriophage M13mpl 8. This template was primed with a single DNA 57mer oligonucleotide and the Pol III enzyme was tested for conversion of this template to a double strand circular form (RFII). The reaction was supplimented with recombinant T.th. β produced in E. coli. This assay is summarized in the scheme at the top of Fig. 26. M 13mpl 8 ssDNA was phenol extracted from phage purified as described (Turner and O'Donnell. 1995). M13mpl 8 ssDNA was primed with a 57mer DNA oligomer synthesized by Oligos etc. The replication assays contained 73 ng singly primed M13mpl 8 ssDNA and 100 ng T.th. β subunit in a 25 μl reaction containing 20 mM Tris-HCl (pH 7.5). 8 mM MgC . 40 μg/ml BSA, 0.1 mM EDTA. 4% glycerol. 0.5 mM ATP, 60 μM each of dCTP, dGTP, dATP and 20 μM α-³²P-TTP (specific activity 2.000-4,000 cpm/pmol). Either T.th. Pol III from the Heparin. peak 1 (HEP.P 1 ; 5 μl, 0.21 units where 1 unit equals 1 pmol nucleotide incoφorated in 1 min.) or a non-Pol III from the Heparin peak 2 (HEP.P2; 5 μl, 2.6 units) were added to the reaction. Reactions were shifted to 60°C for 5 min., and then DNA synthesis was quenched upon adding 25 μl of 1% SDS. 40 mM EDTA. One half of the reaction was analyzed in a 0.8% native agarose gel. and the other half was quantitated using DE81 paper as described (Studwell and O'Donnell. 1990).

The results of the assay are shown in Fig. 26. Lane 1 is the result obtained using the T.th. Pol III (HEP.P 1 ) which was capable of extending the primer around the ssDNA circle to form RFII. Lane 2 shows the result of using the non-Pol III (HEP.P2) which was not capable of this extension and produced only incomplete DNA products (the result shown included 0.8 μg E. coli SSB which did not increase the chain length of the product. In the absence of SSB. the same product was observed, although the band contained more counts. The greater amount of total synthesis observed in lane 2 is due to the build up of immature products in a small region of the gel. The presence of immature products in lane 1 is likely due to a contaminating polymerase in the preparation that can not convert the single primer to the full length RFII form. Alternatively, the presence of incomplete products in lane 1 (Pol III type enzyme) is due to secondary structure in the DNA which causes the Pol III to pause. In this case it may be presumed that performing the reaction at higher temperature could remove the secondary structure barrier. Alternatively, SSB (single strand binding protein) could be added to the assay (although T.th. SSB would be needed since addition of E. coli SSB was tried and did not alter the quality of the product profile). Generally. SSB is needed to remove secondary structure elements from ssDNA at 37 °C for complete extension of primers by mesophilic Pol Ill-type enzymes.

The assay described above was performed at 60 °C. The T.th. Pol III HEP.P 1 gained activity as the temperature was increased from 37 °C to 60 °C. as expected for an enzyme from a thermophilic source. The E. coli Pol III lost activity at 60°C compared to 37°C. as expected for an enzyme from a mesophilic source.

The following is a list of documents related to the above disclosure and particularly to the experimental procedures and discussions. The documents should be considered as incoφorated by reference in their entirety.

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Chen. M.. Pan. Z.-Q., and Hurwitz, J. (1992) Studies of the cloned 37-kDa subunit of activator 1 (replication factor C) of HeLa cells. Proc. Natl. Acad. Sci. USA 521 1-5215.

Cullman. G.. Fien. K.. Kobayashi. R.. and Stillman. B. (1995) Characterization of the five replication factor C genes of Saccharomyces cerevesiae. Mol. and Cell. Biol. 15 0:4661-4671. Flower. A.M. and McHenry. CS. (1990) The γ subunit of DNA polymerase III holoenzyme of Escherichia coli is produced by ribosomal frameshifting. Proc. Natl. Acad. Sci. USA 87:3713-3717.

Guenther, B.D. ( 1996) Structural studies on the DNA replication apparatus: X-ray crystal structure of the δ' subunit of Escherichia coli DNA Pol III. Ph.D. Thesis. Rockefeller University.

Guibus, J.M., Kelman. Z., Hurwitz, J., O^'Donnell, M., and Kuriyan. J. (1996) Structure of the C-terminal region of p21wafl/cipl complexed with human PCNA. Cell 87:297-306.

Jacks, T., Madhami. H.D., Masiarz, F.R.. and Varmus, H.E. (1988) Signals for ribosomal frameshifting in the Rous sarcoma virus gag-pol region. Cell 55:447-458.

Kelman, Z.. and O'Donnell, M. (1994) DNA replication: enzymology and mechanisms. Current Opinion is Genetics and Development 4: 185-195.

Kong. X. -P., Onrust. R., O'Donnell, M., and Kuriyan, J. (1992). Three dimensional structure of the β subunit of Escherichia coli DNA polymerase III holoenzyme: a sliding DNA clamp. Cell 69:425-437.

Kornberg, A., and Baker, T. (1992). DNA Replication, second edition. (New York:W. H. Freeman and Company), pp. 165-194.

Krishna, T.S.. Kong. X.-P., Gary, S. , Burgers, P.M., and Kuriyan, J. ( 1994 ) Crystal structure of the eukaryotic DNA polymerase processivity factor PCNA. Cell 79 (7), 1233-1243. Kuriyan. J. and O'Donnell. M. (1993) Sliding clamps of DNA polymerases. J. Mol. Biol. 234:915-925.

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This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The present disclosure is therefore to be considered as in all respects illustrative and not restrictive, the scope of the invention being indicated by the appended claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

Claims

WHAT IS CLAIMED IS:

1. A DNA Polymerase Ill-type enzyme found in a thermophilic bacterium which exhibits the following characteristics: the ability to extend a primer over a long stretch of ssDNA at elevated temperature; the ability to be stimulated by a cognate sliding clamp of the type that is assembled on DNA by a 'clamp' loader (e.g. ╬│ complex); contains associated clamp loading sub-units that show DNA stimulated ATPase activity at elevated temperature and/or ionic strength; and an accessory protein with DNA polymerase-associated 3'-5' exonuclease activity.

2. The DNA Polymerase Ill-type enzyme according to Claim 1 which is isolated from a thermophilic bacterium selected from Thermus and Thermatoga species.

3. The DNA Polymerase Ill-type enzyme according to Claim 2. wherein the thermophilic bacterium comprises a member of the Thermus species.

A. The DNA Polymerase Ill-type enzyme according to Claim 3, wherein the thermophilic bacterium comprises Thermus thermophilus.

5. The DNA Polymerase Ill-type enzyme according to Claim 1, which comprises at least one of the following: A. a ╬│ subunit having an amino acid sequence selected from the formula set forth in SEQ ID NOS:4 and 5; B. a ╧ä subunit having an amino acid sequence corresponding to the formula set forth in SEQ ID NO:2; C. a e subunit having an amino acid sequence corresponding to the formula set forth in SEQ ID NO:95; D. a subunit comprising an amino acid sequence corresponding to the formula set forth in SEQ ID NO: 87; E. a ╬▓ subunit having an amino acid sequence corresponding to the formula set forth in SEQ ID NO: 107; and variants, including allelic variants, muteins. analogs and fragments of any of subparts (A) through (E), and combinations thereof, capable of functioning in DNA amplification and sequencing.

6. The DNA Polymerase Ill-type enzyme of Claim 1, which includes a ╬│ sub-unit which exhibits a frameshift as great as -2.

7. An isolated polynucleotide encoding a ╧ä subunit of a Thermus thermophilus DNA polymerase Ill-type enzyme, wherein said ╧ä subunit has a molecular weight of about 58.000 daltons as determined by SDS-PAGE under non-reducing conditions.

8. An isolated polynucleotide according to Claim 7, wherein said amino acid residue sequence is represented by the formula shown in SEQ ID NO:2. analogs thereof, muteins thereof, alleles thereof, and active fragments thereof.

9. An isolated polynucleotide according to Claim 7, wherein the polynucleotide sequence is the polynucleotide sequence of positions 132 to 1713 of SEQ ID NO: l, conserved variants thereof, analogs thereof, active fragments thereof, and combinations thereof.

10. An isolated polynucleotide according to Claim 7, wherein the polynucleotide is the polynucleotide sequence of positions 1 to 2027 of SEQ ID NO: l, conserved variants thereof, analogs thereof, active fragments thereof, and combinations thereof.

1 1. An isolated nucleic acid molecule encoding at least a single subunit of a DNA polymerase Ill-type enzyme found in a thermophilic bacterium, which nucleic acid molecule is selected from the group consisting of: dnaX:

B. dnaQ:

C. dnaE:

D. dnaN:

F. variants, including conserved variants, analogs and fragments of any of subparts (A) through (D), and combinations thereof, capable of functioning in DNA amplification and sequencing.

12. The isolated nucleic acid molecule according to Claim 1 1, wherein said nucleic acid molecule comprises dnaX.

13. The isolated nucleic acid molecule according to Claim 1 1. wherein said nucleic acid molecule comprises dnaQ.

14. An isolated nucleic acid molecule associated with a DNA polymerase Ill-type enzyme found in a thermophilic bacterium, wherein said nucleic acid molecule comprises dnaA.

15. The isolated nucleic acid molecule according to Claim 1 1, wherein said nucleic acid molecule comprises dnaN.

16. A subunit of a DNA polymerase Ill-type enzyme found in a thermophilic bacterium, which subunit has an amino acid sequence selected from the group consisting of SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:2: SEQ ID NO:95: SEQ ID NO:87; SEQ ID NO: 107; muteins thereof; alleles thereof; analogs thereof; active fragments thereof; and combinations thereof.

17. The subunit of Claim 16. wherein said subunit has an amino acid sequence selected from SEQ ID NO:4 and SEQ ID NO:5. and comprises the ╬│ subunit of a Thermus thermophilus DNA polymerase Ill-type enzyme.

18. The subunit of Claim 16. wherein said subunit has an amino acid sequence set forth in SEQ ID NO:2. and comprises the ╧ä subunit of a Thermus thermophilus DNA polymerase Ill-type enzyme.

19. The subunit of Claim 16, wherein said subunit has an amino acid sequence set forth in SEQ ID NO:95, and comprises the e subunit of a Thermus thermophilus DNA polymerase Ill-type enzyme.

20. The subunit of Claim 16, wherein said subunit has an amino acid sequence set forth in SEQ ID NO: 87, and comprises the ╬▒ subunit of a Thermus thermophilus DNA polymerase Ill-type enzyme.

21. The subunit of Claim 16. wherein said subunit has an amino acid sequence set forth in SEQ ID NO: 107, and comprises the ╬▓ subunit of a Thermus thermophilus DNA polymerase Ill-type enzyme.

22. A vector comprising an isolated nucleic acid molecule taken from Claim 11.

23. A vector comprising the isolated nucleic acid molecule of Claim 12.

24. A vector comprising the isolated nucleic acid molecule of Claim 13.

25. A vector comprising the isolated nucleic acid molecule of Claim 14.

26. A vector comprising the isolated nucleic acid molecule of Claim 15.

27. A vector selected from pETdnaX and pETdnaN.

28. A host cell comprising at least one of the vectors of Claim 22.

29. The host cell according to Claim 28. wherein the host cell is a prokaryotic cell.

30. A host cell comprising a vector according to Claim 23.

31. A host cell comprising a vector according to Claim 24.

32. A host cell comprising a vector according to Claim 25.

33. A host cell comprising a vector according to Claim 26.

34. The host cell according to Claim 30, wherein the host cell is a prokaryotic cell.

35. The host cell according to Claim 31. wherein the host cell is a prokaryotic cell.

36. The host cell according to Claim 32, wherein the host cell is a prokaryotic cell.

37. The host cell according to Claim 33, wherein the host cell is a prokaryotic cell.

38. An isolated DNA which codes for a recombinant DNA polymerase Ill-type enzyme, or subunit thereof, from a thermophilic bacterium, consisting essentially of a DNA fragment which hybridizes in a Southern blot to an isolated DNA fragment selected from the group consisting of the DNA fragments defined in SEQ ID NO:6 and the DNA fragments defined in SEQ ID NO:8. wherein hybridization is conducted under the following conditions: a) hybridization: 1% crystalline BSA (fraction V) (Sigma). 1 mM EDTA. 0.5 M NaHP0 (pH 7.2), 7% SDS at 65┬░C for 12 hours and; b) wash: 5 x 20 minutes with wash buffer consisting of 0.5% BSA, fraction V). ImM Na_:EDTA. 40 mM NaHP0₄ (pH 7.2), and 5% SDS.

39. A cloning vector comprising the isolated DNA of Claim 38.

40. A host cell transformed by the vector of Claim 39.

41. A method for producing a recombinant thermostable DNA polymerase Ill-type enzyme, or subunit therof, from a thermophilic bacterium comprising culturing a host cell transformed with the vector of Claim 39 under conditions suitable for the expression of said DNA polymerase Ill-type enzyme or said subunit.

42. A DNA probe which hybridizes to the DNA sequence coding for the thermostable DNA polymerase Ill-type enzyme, or subunit therof, of Claim 38. wherein the DNA probe is selected from the group consisting of SEQ ID NO:6 and SEQ ID NO:8.

43. A method for isolating a target DNA fragment consisting essentially of a DNA coding for a thermostable DNA polymerase Ill-type enzyme, or subunit therof, from a thermophilic bacterium comprising the steps of: (a) forming a genomic library from the bacterium; (b) transforming or transfecting an appropriate host cell with the library of step (a); (c) contacting DNA from the transformed or transfected host cell with a DNA probe which hybridizes to a DNA fragment selected from the group consisting of the DNA fragments defined in SEQ ID NO:6 and the DNA fragments defined in SEQ ID NO:8. wherein hybridization is conducted under the following conditions: i) hybridization: 1% crystalline BSA (fraction V) (Sigma). 1 mM EDTA. 0.5 M NaHP04 (pH 7.2). 7% SDS at 65 ┬░C for 12 hours and; ii) wash: 5 x 20 minutes with wash buffer consisting of 0.5% BSA, fraction V). ImM Na₂EDTA. 40 mM NaHPO₄ (pH 7.2), and 5% SDS: (d) assaying the transformed or transfected cell of step (c) which hybridizes to the DNA probe for DNA polymerase Ill-type activity; and (e) isolating a target DNA fragment which codes for the thermostable DNA polymerase Ill-type enzyme or subunit therof.

44. An isolated DNA molecule encoding a protein subunit of DNA polymerase Ill-type enzyme from a thermophilic bacterium wherein the subunit group is selected from the group consisting of ╧ä, ╬│ at a -1 frameshift. ╬│ at a -2 frameshift. e, , and ╬▓.

45. The isolated DNA molecule according to Claim 44..wherein the subunit is ╧ä and has a molecular weight of 58 kD.

46. The isolated DNA molecule according to Claim 45. wherein the protein has an amino acid sequence corresponding to SEQ ID NO:2.

47. The isolated DNA molecule according to Claim 45, wherein the DNA molecule has a nucleotide sequence corresponding to SEQ ID NO:3.

48. The isolated DNA molecule according to Claim 44, wherein the subunit is ╬│ at a -1 frameshift. and has a molecular weight of 50.8 kD.

49. The isolated DNA molecule according to Claim 46. wherein the protein has an amino acid sequence corresponding to SEQ ID NO:4.

50. The isolated DNA molecule according to Claim 44. wherein the subunit is ╬│ at a -2 frameshift. and has a molecular weight of 49.8 kD.

51. The isolated DNA molecule according to Claim 47. wherein the protein has an amino acid sequence corresponding to SEQ ID NO: 5.

52. An expression system comprising an isolated nucleic acid molecule according to Claim 1 1.

53. The expression system according to Claim 52, wherein the protein corresponds to ╧ä and has an amino acid sequence corresponding to SEQ ID NO:2.

54. The expression system according to Claim 53, wherein the DNA molecule has a nucleotide sequence corresponding to SEQ ID NO:3.

55. The expression system according to Claim 52, wherein the protein corresponds to the e subunit and has an amino acid sequence corresponding to SEQ ID NO:95.

56. The expression system according to Claim 55, wherein said subunit has a nucleotide sequence corresponding to SEQ ID NO:94.

57. The expression system according to Claim 52, wherein the protein corresponds to the subunit and has an amino acid sequence corresponding to SEQ ID NO:87.

58. The expression system according to Claim 57, wherein said subunit has a nucleotide sequence corresponding to SEQ ID NO:86.

59. The expression system according to Claim 52, wherein the protein corresponds to the ╬▓ subunit and has an amino acid sequence corresponding to SEQ ID NO: 107.

60. The expression system according to Claim 59. wherein said subunit has a nucleotide sequence corresponding to SEQ ID NO: 106.

61. A host cell transformed with a heterologous nucleic acid molecule according to Claim 1 1.

62. The host cell according to Claim 61, wherein the protein has an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5 SEQ ID NO:87. SEQ ID NO:95, and SEQ ID NO: 107.

63. The host cell according to Claim 61, wherein the nucleic acid molecule has a nucleotide sequence selected from the group consisting of SEQ ID NO: 1. SEQ ID NO:3. SEQ ID NO:86. SEQ ID NO:94. and SEQ ID NO:106.

64. A DNA probe which hybridizes to the DNA sequence coding for the thermostable DNA polymerase Ill-type enzyme of Claim 1 , wherein the DNA probe is selected from the group consisting of the oligonucleotide defined in SEQ ID NO:6; the oligonucleotide defined in SEQ ID NO:8; the oligonucleotide defined in SEQ ID NO: 10; the oligonucleotide defined in SEQ ID NO: 1 1 ; the oligonucleotide defined in SEQ ID NO:12: the oligonucleotide defined in SEQ ID NO: 13; the oligonucleotide defined in SEQ ID NO: 14; the oligonucleotide defined in SEQ ID NO: 15, and the oligonucleotide defined in SEQ ID NO: 16.

65. A method for isolating a target DNA fragment consisting essentially of a DNA coding for a thermostable DNA polymerase Ill-type enzyme, or subunit therof, from a thermophilic bacterium comprising the steps of: (a) forming a genomic library from the bacterium; (b) transforming or transfecting an appropriate host cell with the library of step (a); (c) contacting DNA from the transformed or transfected host cell with a DNA probe of Claim 61 , wherein hybridization is conducted under the following conditions: i) hybridization: 1% crystalline BSA (fraction V) (Sigma). 1 mM EDTA, 0.5 M NaHP0 (pH 7.2), 7% SDS at 65 ┬░C for 12 hours and; ii) wash: 5 x 20 minutes with wash buffer consisting of 0.5% BSA, fraction V). 1 mM Na_:EDTA. 40 mM NaHP0₄ (pH 7.2), and 5% SDS: (d) assaying the transformed or transfected cell of step (c) which hybridizes to the DNA probe for DNA polymerase activity; and (e) isolating a target DNA fragment which codes for the thermostable DNA polymerase Ill-type enzyme or subunit therof.

66. A method for amplifying a nucleic acid molecule, said method comprising contacting said nucleic acid molecule with a composition comprising the DNA polymerase Ill-type enzyme, or subunit thereof, of Claim 1.

67. A DNA molecule amplified by the method of Claim 66.

68. A method of preparing a recombinant vector comprising inserting a nucleic acid molecule taken from Claim 1 1 into a vector.

69. The method of Claim 68, wherein said vector is an expression vector.

70. A recombinant vector made according to the method of Claim 68.

71. A method of making a recombinant host cell, comprising inserting the nucleic acid molecule of Claim 11 into a host cell.

72. The method of Claim 71, wherein said host cell is a bacterial cell, a yeast cell or an animal cell.

73. A kit for amplifying a nucleic acid molecule comprising a carrier and at least two containers, wherein at least the DNA polymerase Ill-type enzyme of Claim 1 is disposed in a first container, and the second container holds other reagent necessary or useful for amplifying said nucleic acid molecule.