JP2003506089A - Chimeric protein - Google Patents

Abstract

(57) Abstract: The present invention provides a) a first domain having a nucleic acid synthesis activity, and b) an interaction mediating sequence, wherein the interaction mediating sequence is a complex of the nucleic acid synthesizing activity and a sliding protein. This complex is directed to a recombinant chimeric protein comprising nucleic acid synthesis activity and / or a complex that differs from the complex that gliding proteins form with their natural interacting partners.

Description

Detailed Description of the Invention

The present invention provides a recombinant chimeric protein having nucleic acid synthesis activity, a complex containing these proteins, nucleic acids encoding these proteins, vectors and cells containing these proteins, antibodies against these, It relates to the application of proteins, kits containing them and methods for nucleic acid extension, amplification, reverse transcription, sequencing and labeling.

Many proteins exist in cells as part of functional multimeric complexes rather than as monomers. Examples of such complexes have been described in almost all areas of cell biology (eg, transcription, translation, replication, cytoskeleton, signaling, mRNA processing).

The interaction or binding of one protein with another, hereafter referred to as a donor or acceptor protein, involves the specific binding or interaction of a folded protein with its partner at the protein surface. It is performed by certain amino acids or amino acid sequences that are responsible for the action. These amino acids or such amino acid sequences are hereinafter referred to as "interaction carrying sequences" or interaction mediating sequences. Amino acids involved in the formation of interactions are not necessarily immediately adjacent to each other in the primary amino acid sequence, but rather in positions within the amino acid sequence of a protein or peptide that are conserved to a greater or lesser extent. It is known to the person skilled in the art that it is the cause of the action. In the description below,
When referring to the determination of sequences that mediate or carry out an interaction, this is intended to mean both sides as described above. In this context, a donor protein is understood as a protein that interacts with and optionally binds another protein. Similarly, an acceptor protein is understood to be a protein that can interact with and bind to another protein.

There are several methods for determining the interaction-performing sequence of a protein-protein interaction site. These include (i) determination of the three-dimensional structure of the complex of donor and / or acceptor proteins by X-ray structural analysis, or (ii) NMR methods. Another method is to (iii) use recombinant protein to reconstitute the binding of the complex in vitro and determine the sequences that perform the interaction by specifically altering the donor or acceptor proteins. is there. Such specific changes include, for example, mutations of individual amino acids that form alanine (alanine scanning) or removal of sequences in the protein. If an amino acid or sequence change or deletion leads to an interaction or loss of avidity, it is part of the sequence performing the interaction, and vice versa. If it does not lead to a loss of binding activity, it is not part of the interaction facilitating sequence. The interaction performance sequence can be defined in this way.

[0005] Another method for determining protein interaction performance sequences is based on the use of a two-hybrid system, also abbreviated hereinafter as "Y2H". The Y2H system is based on the expression of proteins with detectable activity (eg the enzyme dihydrofolate reductase) as two non-covalently linked moieties. This protein is inactive when the two parts are not spatially close to each other. Two proteins to be investigated, known as donor proteins and acceptor proteins, or two proteins to be studied in this regard, which are known to be able to interact with each other and thus form a complex, have detectable activity. Protein having
For example, dihydrofolate reductase), each fused to one of the two moieties to form and express a fusion protein, resulting in the formation of two fusion proteins. If the fused donor protein binds to the fused acceptor protein, the two halves of the detectable protein (eg, dihydrofolate reductase) are spatially proximal. This restores the activity of the protein and makes it detectable. Suitable proteins with detectable activity are enzymes (
Dihydrofolate reductase, β-galactosidase), signal transduction protein (
Saccharomyces cerevisiae-derived Cdc25) or transcription activator (Gal4, LexA-VP16). The determination of the binding region using the two-hybrid system is based on the same considerations as the in vitro rearrangement of binding: if alteration or removal of an amino acid or sequence leads to loss of binding activity, it is If it is part, and conversely, a change or removal of a sequence does not lead to a loss of binding activity, then it is not part of the interaction carrying sequence.

[0006] Furthermore, interaction performance sequences can be elucidated by examining the interactions of multiple fragments of a protein and determining which part of the protein is always present in the interacting fragments. it can. This region, which is always present, is the interaction performance sequence. Individual amino acids can be altered by targeted mutagenesis to identify those amino acids that are essential for the interaction or binding in the interaction performance sequence. Loss or enhancement of binding activity indicates that these positions are directly involved in the interaction.

Particularly preferred complexes for in vitro applications include thermostable complexes of prokaryotic and eukaryotic replicators, which often contain a polymerase as a key enzymatic activity.

As already mentioned, in vivo interactions between proteins play a major role in the replication of nucleic acids in eg biological systems. That is, a highly procedural replication mechanism is known, on the one hand the cellular machinery and, on the other hand, the replication machinery present in bacteriophage T4 and T7.

The duplication device is composed of many elements. Among these are: a) a protein with polymerase activity, b) a protein involved in the formation of the glaze structure (the function of the glaze structure, among other things, links the polymerase activity to its template, stabilizes the binding, and thus Changing the dissociation constant of the polymerase-nucleic acid complex), c) a protein that loads this glaze onto the template, d) a protein that stabilizes the template, and, e) optionally induces the polymerase on the template. Proteins which include

A protein having polymerase activity is here understood in particular to be a protein capable of attaching one or several nucleotides or nucleosides to one nucleotide or nucleoside or a polynucleotide or polynucleoside. To be done. In each of the above cases, these may be ribonucleotides / ribonucleosides or deoxynucleotides / deoxynucleosides or polymers thereof. Thus, these proteins include RNA polymerase in addition to DNA polymerase, regardless of whether a template is required for the polymerase reaction of the protein. Therefore, these proteins having polymerase activity are also proteins having nucleic acid synthesis activity. These also include extension proteins known in the prior art. An extension protein as used herein is also understood as a protein or complex having polymerase activity that has at least one or more of the following properties: RNA as template, DNA as template, RNA synthesized, Synthesize DNA, 5'-3 'exonuclease activity or 3'-5' exonuclease activity, strand displacement activity and processivity or non-processivity.

DNA polymerases belong to a group of enzymes that use single-stranded DNA as a template for the synthesis of complementary DNA strands. These enzymes play a major role in nucleic acid metabolism, including DNA replication, repair and recombination processes. DNA polymerases have been identified in all cell organisms, from bacterial cells to human cells, many viruses and bacteriophages (Kornberg, A. and Baker, TA (1991) DNA.
Replication WH Freeman, New York, NY). Prokaryotes and eubacteria are usually combined into a prokaryotic population, which is an organism that does not have a true cell nucleus, as opposed to a eukaryote, which is an organism that has a true cell nucleus. A feature common to many polymerases from different organisms is often amino acid sequence similarity and structural similarity (Wang, J., Sattar, AKMA; Wang, CC, Karam, JD, Konigsberg, WH and Steitz. , TA (1997) Polα family replication DNA from bacteriophage RB69.
Crystal structure of polymerase, Cell 89, 1087-1099). Human beings have many DNA
Although they have dependent polymerases, not all are responsible for DNA replication, some also perform DNA repair. Replicating DNA polymerases are usually composed of protein complexes with several units that replicate in vivo the chromosomes of the cell organism and the virus. The collective nature of these replicative polymerases is generally a high degree of processivity, meaning the ability to polymerize thousands of nucleotides without dissociating from the DNA template (Kornberg, A. and Baker, TA ( 1
991) DNA Replication, WH Freeman, New York, NY).

DNA polymerases are characterized in particular by two properties: the rate of extension, the number of nucleotides per second that can be incorporated into the growing DNA strand, and the dissociation constant. If the polymerase dissociates from the strand again after each step where one nucleotide is incorporated into the growing strand (i.e., one extension step per binding event), then the processivity is given by the number 1. And, this polymerase is not processive. If the polymerase remains bound to the strand during the uptake of the nucleic acid many times, the extension or replication mode, and thus also the polymerase, is called processive and the number can reach thousands. Methods
in Enzymology 262, DNA replication, edited by JL Campbell, Academic Press 1995, pp.
See also 270-280).

The proteins mentioned as b) form either open or closed, eg cyclic or semi-cyclic structures. Such a structure may be formed by one or several proteins. One such protein species may have polymerase activity.

The proteins responsible for the formation of these structures are hereinafter referred to as “slip glaze proteins” or “glare proteins”, unless they have polymerase activity.

Although the genome organization of eukaryotes and archaebacteria is quite different, the replication machinery of archaebacteria is similar to that of eukaryotes, and the cell structure of eubacteria is similar to that of archaebacteria. (Edgell, DR and Doolittle, WF (1997)
, Archebacteria and sources of DNA replication proteins, Cell 89, 995-998).

The glaze is often linked to the extension protein via one or several other proteins, in other words it is coupled to the extension protein. Such a coupling protein is hereinafter referred to as a coupling protein,
Here, the coupling can occur via a number of coupling proteins.

The three-dimensional structure of various gliding proteins has already been determined. The overall structures of these glazes are very similar; photographs of the entire cyclic protein structure of PCNA, the β subunit, and the gp45 ring overlap exactly when placed on top of each other (Kelman,
Z. and O'Donnel, M. (1995) Structural and functional similarities of prokaryotic and eukaryotic sliding glazes, Nucleic Acids Res. 23, 3613-3620). Each circle has comparable size and a central opening large enough to enclose a double-stranded DNA, ie, a DNA double strand composed of two complementary DNA strands.

The gliding glaze cannot position itself around the DNA in vivo and this D
Must be kept around the NA. In prokaryotes and eukaryotes, such protein complexes are composed of multiple subunits. The protein complex recognizes the 3'end of the primer in the "primer-template duplex", which extends by incorporation of nucleotides to form a longer duplex, and slips glides around this DNA. Position it.

In the case of bacteriophage T7, it has the same purpose, namely
DNA synthesis is achieved by protein complexes with different structures. This phage expresses its catalytic polymerase T7 polymerase, the gene product of the fifth gene, which is a protein derived from the host Escherichia coli,
That is, it binds to thioredoxin and enables highly processive DNA replication as a replicase. In this case, there is also glaze formation, but this glaze does not have the same structure as that of eukaryotic PCNA, for example.

It is often necessary, for example, as in the case of human polymerase δ, that the protein (coupling protein) makes a bond between the catalytically active part of the polymerase and the processivity factor. A processivity factor is a compound or molecule that affects, and preferably increases the processivity of a polymerase. Sliding glaze protein is an example of a processivity factor. In humans this coupling protein is a small subunit of δ polymerase (Zhang, S.-J., Zeng, X.-U., Zhang, P., Toom
ey, NL, Chuang, RY, Chang, L.-S. and Lee, MYWT (1994). The amino-terminal conserved region of DNA polymerase δ is involved in the proliferation of nuclear antigen binding, J. Bi
ol. Chem. 270, 7988-7992). However, in the case of T7 polymerase, the processivity factor binds directly to the catalytic unit of the polymerase without involvement of the coupling protein.

In vitro applications of proteins with nucleic acid synthesis activity or extension proteins, such as polymerases, are widespread in the prior art, for example for polymerase chain reaction (PCR), nucleic acid sequencing or reverse transcription.

For most in vitro applications, such as PCR or sequencing processes, processivity is a desirable property, provided that the prior art thermostable enzymes used in these reactions have this property to a small extent. I just have to.

US Pat. Nos. 4,683,195, 4,800,195 and 4,683,202 describe the application of such thermostable DNA polymerases to the polymerase chain reaction (PCR). PC
In R, DNA is de novo synthesized using primers, templates (also called matrices), nucleotides, DNA polymerase, suitable buffers and suitable reaction conditions.
Preferably, a thermostable polymerase that survives the cyclic thermal melting of the DNA strand is used for this PCR. Therefore, Taq DNA polymerase is often used (US Patent No.
4965188). However, the processivity of Taq DNA polymerase is relatively lower than that of T7 polymerase.

DNA polymerase is also used for DNA sequencing (Sanger et al., Proc. Natl. Acad. Sci.
, USA 74: 5463-5467 (1997)). One of the polymerases that can be used for this is, for example,
Taq polymerase (U.S. Pat. No. 5,075,216) or Termotoga neapolitana described above.
(Thermotoga neapolitana) -derived polymerase (WO 96/10640) or other thermostable polymerases. Recent methods combine exponential amplification and sequencing of DNA fragments into a single step, so that genomic DNA can be sequenced directly. One of these methods, the so-called DEXAS method (Nucleic Acid
s Res. 1997 May 15:25 (10): 2032-2034 Direct DNA sequencing from total genomic DNA, Ki
lger C, Paabo S, Biol Chem. 1997 Feb; 378 (2): 99-105, direct exponential amplification and sequencing of genomic DNA (DEXAS), Kilger C, Paabo S and DE19653439.9 and DE19653494.1. ) Is a polymerase with a low ability to discriminate dideoxynucleotides (ddNTPs) from deoxynucleotides (dNTPs) in order to obtain complete, fragment-fragmented, sequence-specific DNA ladders adjacent to the primers in a few cycles, as well as the reaction. A buffer, preferably two primers that are not present in equimolar amounts, and the above nucleotides are used. A further development of this method involves the use of a polymerase mixture in which one of the two polymerases discriminates between ddNTPs and dNTPs, and the other has reduced discriminating power (Nucleic Acids Res. 1997 May 15; 25 ( 10) 2032-2034
Direct DNA sequencing from total genomic DNA, Kinger C, Paabo S).

DNA polymerase is also used for reverse transcription of RNA into DNA. In this case, RNA acts as a template and the polymerase synthesizes a complementary DNA strand. For example, a thermostable DNA polymerase (US Pat. No. 5,322,770) from the organism Thermus thermusphilis (Tth) is used in this case.

Some polymerases also have “proofreading” activity, ie 3′-5 ′.
Has exonuclease activity. This property is particularly desirable when the product to be synthesized should be produced with a low rate of nucleotide incorporation errors. An example is the polymerase from the organism Pyrococcus wosei.

The vast majority of the above extension proteins used in the above-mentioned applications are actually enzymes that are thought to be primarily involved in DNA repair, rather than in vivo replicative enzymes, which makes them relatively less processive. That's a small reason. In addition, each organism has multiple polymerases with many properties.

As mentioned above, such an extension protein has, for example, the following properties: use of RNA as template, use of DNA as template, RNA synthesis, DNA synthesis, 5′-3 ′.
Direction exonuclease activity and 3'-5 'direction exonuclease activity,
Strand displacement activity, processivity or non-processivity or heat stability or heat sensitivity. However, in vivo, protein complexes often combine one or several of these properties. Among other things, as noted above, replication complexes are often present in vivo with increased processivity due to the presence of gliding proteins.

In addition to the elements of the replication machinery, other DNA-modifying proteins are also often found in vivo in large complexes with other proteins. In such a complex, the DNA-modifying activity, such as the terminal transcriptase activity from the first protein, may be associated with one or several activities introduced into the complex by another protein, such as an exonuclease. Often combined with activity. DNA modifying activity is understood herein as any enzymatic activity which leads to a chemical, physical or structural alteration of the initial nucleic acid. It is sometimes the case that DNA modification activity only occurs when it interacts with at least one additional protein. The DNA modifying activity can also be reduced or enhanced by interaction with at least one additional protein. Thus, in vivo, for example, a protein complex having the sum of the individual activities or a protein complex in which the activity is improved as compared with the individual activities is often formed.

What has been stated above is that in vivo complexes with nucleic acid synthesis activity are often technical,
That is, it has additional properties desirable for in vitro applications, indicating that it exceeds the actual nucleic acid synthesis activity and is contributed by other elements that form the complex.

The direct technical application of such in vivo complexes, for example for the purpose of DNA sequencing, carrying out the polymerase chain reaction or the introduction of labels into nucleic acids, has failed in the past for several reasons. One reason is the lack of knowledge of all factors or individual elements involved in the formation of the complex of interest. Another reason is that composites containing several elements have one or several undesired properties in addition to the desired properties.

An alternative approach to the in vitro use of in vivo complexes is to combine elements of different origin but with the desired properties, thus forming a complex with the desired properties. Such a property may be, for example, higher processivity. An impediment to implementation of this approach is that the individual elements that form the complex that must interact with each other do not interact, or interact only to a very small extent, and thus the desired properties of the complex. Was not possible.

Therefore, it is an object of the present invention to provide a protein having nucleic acid synthesis activity with increased processivity.

A further object of the invention is to provide a method allowing the assembly of such proteins.

Finally, it is an object of the present invention to provide a method for the amplification of nucleic acids, in particular by PCR, which allows longer nucleic acids to be amplified and / or sequenced. Is.

The purpose is to: a) a first domain having nucleic acid synthesis activity, and b) an interaction mediating sequence, wherein the interaction mediating sequence is responsible for the formation of a complex between the nucleic acid synthesis activity and the gliding protein. And this complex is different from the complex with which nucleic acid synthesis activity and / or gliding proteins form with their natural interaction partners].

In a further aspect of the invention, a moiety having nucleic acid synthesis activity, wherein the moiety is derived from a base protein comprising a moiety having nucleic acid synthesis activity and an interaction mediating element, and at least one interaction This object is achieved by a chimeric protein comprising an intermediary element, where this interaction mediation element is different from that of the basic protein.

[0038] In one embodiment, the protein comprises an interaction mediating element of a base protein.

In a further aspect, a chimeric protein comprising a moiety having nucleic acid synthesis activity, wherein the moiety comprises a base protein comprising a moiety having nucleic acid synthesis activity but no interaction mediating moiety, and a derivative from an interaction mediating moiety. , To achieve this goal.

In the chimeric protein according to the present invention, an interaction mediating part can be provided in order to mediate the binding between the nucleic acid synthesizing activity and the factor affecting the rate of synthesis of the nucleic acid synthesizing activity. In a preferred embodiment, the factor is a gliding protein.

In one embodiment, the nucleic acid synthesis activity is intended to include a consensus peptide sequence, which sequence is selected from the group comprising SEQ ID NOs: 1, 2 and 3.

In a further embodiment, the gliding protein can comprise a consensus peptide sequence, which sequence is selected from the group comprising SEQ ID NOs: 4, 5, 6 and 7.

In yet another embodiment, the interaction mediating sequences are SEQ ID NOs: 8, 9, 10, 11 and 12.
Can include a consensus peptide sequence selected from the group comprising In a particularly preferred embodiment, this interaction mediating sequence comprises the consensus peptide sequence according to SEQ ID NO: 8.

In a further embodiment, the interaction mediating sequence is C of a sequence having nucleic acid synthesis activity.
At the end.

In a further embodiment, a linker is located between the interaction mediating sequence and the sequence having nucleic acid activity.

Furthermore, in one embodiment of the chimeric protein according to the present invention, the recombinant chimeric protein may be thermostable.

In one embodiment of the chimeric protein according to the present invention, the protein may have DNA polymerase activity. It is particularly preferred that this protein has 3'-5 'exonuclease activity.

In a further aspect of the chimeric protein according to the invention, the protein has RNA polymerase activity.

In a further aspect of the chimeric protein according to the invention, the protein has reverse transcriptase activity.

In a further preferred embodiment of the chimeric protein according to the invention, the uptake rates of dNTP and ddNTP due to this nucleic acid synthesis activity differ by a factor of less than 5.

[0051]   In a further aspect of the invention, this object is   a) a recombinant chimeric protein according to the present invention, and   b) gliding protein, This is achieved by a complex containing

In a preferred embodiment of the complex according to the present invention, the gliding protein is SEQ ID NO:
It comprises a consensus peptide sequence selected from the group comprising sequences according to 4, 5, 6 and 7.

In a further aspect, the complex further comprises a nucleic acid. In a further aspect, this object is achieved by a nucleic acid encoding a chimeric protein according to the invention, in particular a recombinant protein.

In yet another aspect, this object is achieved by a vector comprising the nucleic acid according to the invention. In a preferred embodiment, this vector is an expression vector.

In a further aspect, this object is achieved by a cell containing the vector according to the invention.

This object is further achieved by the use of the chimeric protein according to the invention for extending a nucleic acid.

This object is also achieved by the use of the chimeric protein according to the invention for amplifying nucleic acids.

This object is also achieved by the use of the chimeric protein according to the invention for reverse transcribing RNA into DNA.

This object is also achieved by the use of the chimeric protein according to the invention for sequencing nucleic acids, especially DNA.

In yet another embodiment, this object is achieved by a kit for the extension and / or amplification and / or reverse transcription and / or sequencing and / or labeling of nucleic acids, in particular a reagent kit, which comprises In several containers, a) a chimeric protein according to the invention, preferably a recombinant protein, and / or b) a complex according to the invention, and c) preferably at least one primer, optionally a buffer. , Nucleotides, cofactors and / or pyrophosphatases.

In a preferred embodiment of this kit, it comprises deoxynucleotides or / and derivatives thereof in addition to substances a) and / or b) for amplifying nucleic acids.

In a further aspect, the kit comprises a DNA having 3'-5 'exonuclease activity.
Contains a polymerase.

In another embodiment, the kit comprises substances according to a) and / or b) having reverse transcriptase activity, and preferably deoxynucleotides and / or derivatives thereof for reverse transcription.

The kit according to the present invention can comprise, in addition to deoxynucleotides or ribonucleotides or / and derivatives thereof, dideoxynucleotides or / and derivatives thereof for sequencing.

In a further aspect, this object is achieved by a method for template-dependent extension of a nucleic acid, wherein the nucleic acid or at least one strand thereof to be extended has at least one under hybridization conditions. Is provided, which is sufficiently complementary to a portion or flanking region of the nucleic acid to be extended,
And this primer is extended by the polymerase in the presence of nucleotides [
Here, the chimeric protein according to the invention is used as a polymerase, preferably a gliding protein is present in this reaction].

In yet another aspect, this object is achieved by a method for the amplification of nucleic acids,
Where at least two primers are added to the nucleic acid to be amplified under hybridization conditions, each of the two primers being complementary to a portion or flanking region of the nucleic acid to be amplified, This primer is then extended by a polymerase in the presence of nucleotides [wherein the chimeric protein according to the invention and especially the recombinant chimeric protein are used as polymerase, preferably a gliding protein is added to this reaction].

In a preferred embodiment, the polymerase chain reaction is performed. In a particularly preferred embodiment, the reaction mixture comprises two DNA polymerases, at least one of which has 3'-5 'exonuclease activity, the 3'-5' exonuclease activity being reacted by the chimeric protein or by an additional polymerase. Add to the mixture.

In yet another embodiment, two chimeric proteins, in particular one protein is DN
There are recombinant chimeric proteins that have A polymerase activity and the other 3'-5 'exonuclease activity.

In the method according to the invention for template-dependent extension, in one embodiment the nucleic acid is sequenced starting from a primer which is complementary to the region flanking the nucleic acid to be sequenced, the method of Sanger Template-dependent extension or reverse transcription can be carried out using deoxynucleotides and dideoxynucleotides or derivatives thereof according to.

In a further aspect of the method according to the invention for template-dependent extension, at least one label can be introduced during the extension of the nucleic acid.

In a particularly preferred embodiment, a reagent selected from the group comprising labeled primers, labeled deoxynucleotides and their derivatives, labeled dideoxynucleotides and their derivatives and labeled ribonucleotides and their derivatives is used. .

In yet another embodiment, a single break is made in the phosphodiester bond of the nucleic acid strand and the nucleotide at this break is replaced with a labeled nucleotide with the aid of a polymerase, where the polymerase is the present invention. This object is achieved by a method of labeling nucleic acids by using such chimeric proteins, especially recombinant chimeric proteins.

In yet another embodiment, this purpose is achieved by a method of producing a chimeric protein which comprises a base sequence and a heterologous interaction-carrying sequence and which binds to an interaction partner, or such binding interacts. It is enhanced as a result of the performance sequence, where a) an interaction system containing a protein called a donor protein and a protein called an acceptor protein is used to Determine if it will result in an interaction between the interacting partners, and b) introduce the interaction-carrying sequence into the recipient protein that is different from the donor and acceptor proteins and contains the base sequence.

In one embodiment, the donor and acceptor proteins form a complex that binds with the nucleic acid.

In a further embodiment, the donor and acceptor proteins are polymerase activity, DNA binding activity, RNA binding activity, 5′-3 ′ exonuclease activity, 3
It forms a complex having one activity selected from the group including a'-5 'exonuclease activity and a ligase activity.

In a preferred embodiment, the donor protein is selected from the group comprising extension proteins, gliding proteins, gliding loader proteins and coupling proteins.

In a further aspect, the acceptor protein is selected from the group comprising elongation proteins, gliding proteins, gliding loader proteins and coupling proteins.

In yet another embodiment, the recipient protein is selected from the group comprising elongation protein, gliding protein, gliding loader protein and coupling protein.

In one embodiment of the method according to the present invention, step a) is repeated several times to determine a consensus sequence from the interaction performance sequences determined in this way which represents the interaction performance sequence and this is performed. In b), the interaction performance sequence can be introduced into a recipient protein that is different from the donor protein and the acceptor protein and contains a base sequence.

In a further aspect, this object is achieved by a chimeric protein obtainable by the method according to the invention. In a preferred embodiment, this base sequence is a part of the amino acid sequence of a protein selected from the group consisting of extension proteins, gliding proteins, gliding loader proteins and coupling proteins.

Finally, this object is achieved by an in vitro complex for template-dependent extension of nucleic acids, including gliding proteins and extension proteins, wherein at least one of the proteins according to the invention It is a chimeric protein. In this regard, embodiments in which the composite is thermostable are particularly preferred.

In the context of the present invention, any of the chimeric proteins according to the present invention can be recombinant chimeric proteins.

The object is further provided that the a) first domain having nucleic acid synthesis activity and b) the interaction mediating sequence result in the formation of a complex between the nucleic acid synthesis activity and the gliding protein. Achieved according to the invention by a recombinant chimeric protein comprising an interaction-mediating sequence characterized in that the nucleic acid-synthesizing activity and / or the gliding protein are different from the complex formed by their natural interaction partners. To do.

The recombinant chimeric protein according to the invention thus allows the nucleic acid synthesizing activity to bind, for example, a gliding protein which cannot bind to the nucleic acid synthesizing activity as it naturally occurs. Thus, a natural interaction partner is one that can exist in an organism under normal physiological conditions bound to the nucleic acid synthesis activity.

An object of the present invention is a moiety having nucleic acid synthesis activity, wherein this moiety is derived from a moiety having nucleic acid synthesis activity and a basic protein containing an interaction mediating moiety,
And a at least one interaction-mediating moiety, wherein the interaction-mediating moiety is different from the interaction-mediating moiety of the base protein, and a moiety having nucleic acid synthesis activity, wherein the moiety is a nucleic acid Achieved by a chimeric protein comprising an interaction-mediating moiety, which is derived from a basic protein that comprises a moiety having synthetic activity but not an interaction-mediating moiety.

According to the invention, this object is further achieved by a method for template-dependent extension of a nucleic acid, wherein the nucleic acid to be extended or at least one strand thereof is at least one under hybridisation conditions. One primer is provided, wherein the primer is sufficiently complementary to a part of the nucleic acid to be extended or a flanking region thereof, polymerase extension uses a recombinant chimeric protein according to the invention as a polymerase, It is preferably carried out in the presence of nucleotides, characterized in that a gliding protein is present during this reaction.

“Recombinant” in the sense of the present invention means, for example, that chimeric protein is produced, for example, by genetic engineering (“Gentechnologie, Rompp Basislexikon C
hemie, Georg Thieme Verlag ”1998) or, for example, is understood to mean chemically synthesized.

Other aspects result from the dependent claims. The basis of the present invention is to start with a protein having nucleic acid synthesizing activity, hereinafter referred to as the basic protein, by interacting this protein with factors such as gliding protein that increase the processivity. It is possible to increase the processivity of this protein, or more precisely the activity of nucleic acid synthesis [where the basic protein is not able to interact with this factor as it is, and if it does, it may increase the processivity. Is not connected]. It provides a group of several amino acids, typically in the form of a contiguous amino acid sequence, to a protein having nucleic acid synthesizing activity [this is referred to herein as an interaction mediator or interaction performance sequence]. (And its only consequence is a possible interaction between the protein of interest and a factor that increases processivity). This results in the above-mentioned problems of the prior art,
That is, the problem of lacking compatibility with the desired individual element of the complex having nucleic acid synthesis activity can be overcome.

Without wishing to be limited thereto, this yields at least the following three approaches for the formation of such chimeric proteins with nucleic acid synthesis activity.

The basic proteins that form the basis of chimeric proteins can exist in two basic forms. In the first form, the basic protein contains only nucleic acid synthesis activity and is capable of mediating the interaction between the nucleic acid synthesis activity and the processivity factor, especially in vivo, particularly that interaction leads to increased processivity. It does not include sequences that can be mediated in such a way (also referred to herein as domains).

In a second form, the basic protein is capable of promoting nucleic acid synthesizing activity and, in addition, the interaction of the nucleic acid synthesizing activity with a processivity factor, especially in vivo, in particular that interaction is responsible for the processivity. It includes sequences that can be promoted in a manner that increases them.

The chimeric proteins according to the invention can exist in different basic ways, based on the various forms of the basic protein.

(I) A first embodiment of the chimeric protein uses the first form of the basic protein and attaches to it a sequence that mediates an interaction with a factor that increases the processivity of nucleic acid synthesis activity. I will provide a. The ligation is typically carried out such that the interaction mediating sequence joins the nucleic acid synthesis active sequence and is optionally separated by a linker. Thus, in this first aspect of the chimeric protein, an interaction mediating sequence is added to the base protein.

(Ii) In the second embodiment of the chimeric protein, the second form of the basic protein is used. In this case, the original sequence of the basic protein that mediates the interaction with the factor that increases the nucleic acid synthesis activity is replaced with another sequence that mediates the interaction with the factor that increases the nucleic acid synthesis activity. Replace with. This different sequence can be derived from another gene of the same organism, from the same gene of another organism, or from another gene of another organism, and is thus of different origin in any case.
As a result, such an assembly allows the nucleic acid synthesis activity of the basic protein to interact with factors that increase the processivity of the nucleic acid synthesis activity for the first time or to an increased degree.

(Iii) In the third embodiment of the chimeric protein, the second form of the basic protein is used. In this case, the original sequence of the basic protein that mediates the interaction with the factor that increases the activity of nucleic acid synthesis activity, and another sequence that mediates the interaction with the factor that increases the processivity of nucleic acid synthesis activity. Is added. This different sequence can be derived from another gene of the same organism, from the same gene of another organism, or from another gene of another organism, and is thus of different origin in any case. As a result, such an assembly allows the nucleic acid synthesis activity of the basic protein to interact with factors that increase the processivity of the nucleic acid synthesis activity for the first time or to an increased degree.

It can be said that for all the embodiments of the chimeric proteins mentioned above, the (amino acid) sequences of such chimeric proteins are typically different from the sequence of the basic protein. Further, but not necessarily, the complex formed from the chimeric protein having nucleic acid synthesizing activity and the factor that increases processivity differs from the complex between the base protein and the factor that increases processivity. is doing.

The proteins, polymerases and extension proteins mentioned in this description are all suitable as basic proteins, the description of which is mentioned herein. The same applies to all other components, in particular the components of the replication machinery, such as processivity factors, gliding proteins and interaction mediators or sequences. Finally, the definitions given in the introduction also apply to the content of this part.

Nucleic acid synthesis activity can be derived from many organisms, for example the organism Carboxythermus hydrogenoformans (European patent application EP
0 834 569 A1), or Thermus aquaticus, Thermus caldophilus, Thermus clairophilus
ermus chliarophilus), Thermus filiformis, Thermus flavus, Thermus oshimai, Thermus ruber, Thermus scotod
uctus), Thermus silvanus, Thermus speci
es) ZO5, Thermus species sp. 17, Thermus thermusfils (
Thermus thermusphilus), Therotoga maritima, Therotoga neapolitana, Thermo Sifo Africanus
sipho africanus), Anerocellum thermophilum
), Bacillus caldotenax or Bacillus stearothermophilus.

When referring to a sequence herein, this typically refers to an amino acid sequence. Nucleic acid sequences are usually referred to directly as nucleic acid sequences.

Various nucleic acids encoding the recombinant chimeric proteins of the present invention can be readily determined by those of skill in the art by the genetic code, and can be subsequently synthesized. Similarly, suitable vectors for cloning and expressing recombinant chimeric proteins of the invention and methods for their preferred recombinant production are also well known to those of skill in the art (e.g., Maniatis et al; supra; See).

Antibodies, including monoclonal antibodies, to the recombinant chimeric proteins of the present invention are also well known to those of skill in the art.

The recombinant chimeric proteins according to the invention can be used for eg the polymerase chain reaction, for DNA sequencing, for labeling nucleic acids and for other reactions involving in vivo synthesis of nucleic acids, The nucleic acid can be extended.

Thus, a further subject matter of the present invention is a method for template-dependent extension, wherein this nucleic acid or at least one strand thereof to be extended comprises at least one under hybridization conditions. A primer is provided, wherein the primer is sufficiently complementary to a portion or flanking region of the nucleic acid to be extended, and the primer extension is performed by a polymerase in the presence of nucleotides [wherein the present invention relates to Recombinant chimeric proteins are used as polymerases, and in addition, in a preferred embodiment gliding proteins are present in this reaction or reaction mixture].

The extension is a free 3'- hybridized to the template nucleic acid and available for extension.
Methods for template-dependent extension of nucleic acids initiated by OH-terminated primers are well known to those of skill in the art. In particular the polymerase chain reaction is carried out for this amplification. Double-stranded DNA sequences are commonly used as starting materials intended to amplify certain target regions. Two primers complementary to the region flanking the target sequence on each of the partial strands of this DNA duplex are used for this. However, in order to hybridize the primer, this DNA duplex is first denatured, especially by heat melting.
After hybridization of the primers, extension is carried out with a polymerase, which is then denatured again to separate the newly generated DNA strand from the template strand, but the nucleic acid strand generated in the first step also Because of the extension cycle, it can be used as a template, along with the original template strand, and each of these will be rehybridized with the primer to cause a new extension. This operation is performed in a cycle involving heat denaturation as an intermediate step.

The recombinant chimeric protein according to the invention can also be used for reverse transcription, in which case the protein according to the invention itself has reverse transcriptase activity, or the thermostable in vitro complex is intrinsically reverse-transcribed. An appropriate enzyme having reverse transcriptase activity is further added regardless of whether it has enzyme activity.

The recombinant chimeric protein according to the present invention is also used for reverse transcription of RNA into DNA (
This is preferred according to the invention), in which case the nucleic acid synthesis activity of the protein has an intrinsic reverse transcriptase activity. This reverse transcriptase activity may be the only polymerase activity or may be present with the existing 5'-3 'DNA polymerase activity. A preferred embodiment of the recombinant chimeric protein according to the invention is EP-A 0 834.
Extender proteins derived from the organism Carboxydothermus hydrogenfomans, disclosed in 569.

A further preferred use of the recombinant chimeric proteins according to the invention is to sequence nucleic acids starting from at least one primer which is sufficiently complementary to the part of the nucleic acid to be sequenced, But also perform template dependent extension, or
For RNA sequencing, reverse transcription using deoxynucleotides and dideoxynucleotides is performed according to Sanger's method. Within the framework of this preferred embodiment, the respective derivatives mentioned above are also suitable as deoxynucleotides and dideoxynucleotides. In particular, for the method according to the invention for extending a nucleic acid, it is preferred to label the nucleic acid produced. For this purpose, as already mentioned above, labeled primers and / or labeled deoxynucleotides and / or labeled dideoxynucleotides and / or labeled ribonucleotides or suitable derivatives thereof can be used. .

A further subject matter of the present invention is to insert a discrete break in the phosphodiester bond of the nucleic acid chain, replacing the nucleotide at this break with a labeled nucleotide with the aid of a polymerase [where Using the thermostable in vitro complex according to the invention].

Such a method, commonly referred to as nick translation, allows simple labeling of nucleic acids. All the labeled ribonucleotides or deoxyribonucleotides or their derivatives mentioned above are suitable for this, so long as the polymerase allows them as substrates.

The invention will be further described by the following examples and figures, from which further advantages and aspects result.

Example 1: Use of the yeast two-hybrid system to determine amino acids important for the interaction of replication factors and gliding proteins

The yeast two-hybrid system (Fi) was used to determine the binding regions of gliding proteins (hereinafter referred to as SCP) and replication factors (hereinafter referred to as RF).
elds S., Song O., Nature 1989 Jul 20; 340 (6230): 245-6). Genes of SCP and RF are expressed in the vectors pGBT9 and pGAD424 (CLONTECH Laboratories, Inc.) to produce a fusion protein with the GAL4 DNA binding or activation domain: DNA is the organism Archaeoglobus fulgidus.
lgidus) (DSM No. 4304) by a known method. This microorganism is DSM (“De
utsche Sammlung fur Mikroorganismen "). Next, the open reading frame of this gene was changed to Archeoglobus fulgidus (Archaeo
globus fulgidus) genomic DNA and amplified by PCR. The DNA thus obtained is cloned into the vectors pGBT9 and pGAD424. Other vectors used in the two-hybrid system are also suitable for the methods described below (e.g., these are pAD-GAL4-2.1, pBD-GAL4, pBD-GAL4 Cam, pCMV-AD, pCMV-BD,
pMyr, pSos, pACT2, pAS2-1, pHISi, pLexA, pM, pHISi-1, pB42AD, pVP16, pGA
D10, pGBKT7, placZi, p8op-lacZ, pGAD GH, pGilda, pAD GL, pGADT7, pGBDU,
(Includes pDBLeu, pPC86, pDBTrp, available from CLONTECH Laboratories, Inc.)
. In addition, the modified clone is cloned into the same vector. Modified clones are deletion mutations and mutations that affect individual or some amino acids of SCP and / or RF. The ability of the modified clones to interact with each other in the two-hybrid system is then measured. This allows to determine domains important or essential for the interaction, or even individual amino acid residues.

There are several ways to introduce excision into a gene. One way to make a removal is
To produce a DNA fragment containing the gene for SCP or RF. These fragments are obtained from the appropriate vector by PCR or restriction digests. In addition, genomic DNA from the organism from which these two proteins are derived is used. These various DNA fragments and genomic DNA are sonicated into small pieces and fragments with a length between the full length of the gene and about 100 bases are purified by preparative agarose gel electrophoresis. The ends of the fragment are labeled with the appropriate enzyme (Klenow
, Pwo-polymerase or otherwise) or digested so that both strands have the same length and are therefore blunt. These fragments are incorporated by ligation into the SmaI cleaved (or otherwise linearized and blunted) pGAD424 and pGBT9 vectors, or other suitable vectors. This results in a bank of numerous small fragments of the SCP and RF genes present in both vectors of the two-hybrid system respectively. The DNA of these banks is replicated and purified after transformation with Escherichia coli.

[0114] In the next step, the two banks were paired with each other in a single strain in which the fusion of SCP or RF with the GAL4 DNA binding domain was different from the fusion of RF or SCP with the GAL4 activation domain. If present, introduce into a suitable haploid yeast strain for the two-hybrid system. Then, by pairing the two strains, diploid cells in which the plasmids derived from the two banks are present in the same cell are produced. A preferred strain is PJ69-4 (James P, Halladay J, Craig EA, Genetics 1996 Dec; 144 (4): 1
425-36) or any other strain having a suitable genotype for the two-hybrid system. Cells in which the reporter gene is activated are isolated, plasmid DNA is isolated therefrom by preparation, or the insert is specifically amplified by PCR. R
The region in all clones where the interaction of two fusion proteins including F and SCP occurs.
(Or this is always found in a group of clones),
Determine the binding region (s). This determination is carried out 4 times for each of the genes: once for the bank in the vector with the activation domain (exclusively pGAD424), once for the bank in the vector with the DNA binding domain (exclusively pGTB9), complete with the binding partner. Once for the long clone and once for the gene bank of this fragment.

The experiments described allow to define the minimal binding region available for the assembly of recombinant chimeric proteins with affinity for SCP. To make a more detailed characterization of the binding regions, amino acid mutations are introduced and their effect on the binding properties is tested. This further limits the region and determines the position in the protein involved in binding. Co-preparations also determine the positions involved in binding by introducing random mutations into the binding region and determining its effect on binding. In each case, it is determined whether the mutation destabilizes the protein and thus has an indirect effect, or whether the mutation does not affect the stability of the protein and thus has a direct effect.

The experimental procedure required for this is Maniatis et al. (Molecular Cloning (2nd edition, 3 volumes): Al
aboratory Manual, Cold Spring Harbor Laboratory Press, NY (1989)), Aus
ubel et al. (Current Protocols in Molecular Biology, John Wiley and Sons (1988
)), Abelson, JN, and Simon, MI (eds.) 1991 (Methods in Enzymology, 194).
Volume, Guide to Yeast Genetics and Molecular Biology, Academic press) or
Adams, A. Gottschling, DE Kaiser, CA, and Stearns, T., 1997 (Methods in Y
East Genetics, Cold Spring Harbor Laboratory Press). The operation of the two hybrid system is described in Clontech Company (yeast protocols handbook, PT3024-
Followed the instructions in 1).

Example 2: Additional method for mapping the binding regions of SCP and RF SCP and RF were obtained as recombinant proteins. In each case the two proteins were immobilized on a support and then the other protein was added in unbound form for binding to the immobilized partner. Free or unbound material was washed away and bound protein was eluted, for example by denaturation. The amount of bound protein is a measure of binding (e.g. Anderson D, Koch CA, Gray L, Ellis C. Moran MF
, Pawson T, Science 1990 Nov. 16; 250 (4983): 979-82).

Analysis of deletion mutants of this protein and mutants with modified amino acid sequences allowed mapping of the binding region. Removal can be produced by proteolytic digestion or recombinant expression of the removed gene. In the same way, the binding domain can be determined by co-immunoprecipitation from cell extracts containing the depleted form of the protein. Competition for binding by the peptide provides information about the sequence of the binding region.

A further approach to mapping the binding region is to generate peptides,
In each case the binding of this peptide to other proteins is measured. The peptide sequences may be random (Songyang Z, Prog Biophys Mol.
Biol. 1999; 71 (3-4): 359-72) or based on the amino acid sequence of the protein whose binding region is to be identified (Peptide Scan, Brix J, Rudig.
er S, Bukau B, Shcneider-Mergener J, Pfanner N, J. Biol. Chem. 1999 Jun 4;
274 (23): 16522-30). Such peptides can be produced by chemical synthesis or by other methods such as phage display and used for conjugation. An example is shown in FIG. In this case, the alignment of the various consensus sequences suitable for the production of such peptides was determined.

A further method is to produce antibodies against the epitope of the protein whose binding region is to be identified. If the antibody inhibits binding, the epitope of the antibody partially overlaps the binding region (e.g., Fumagalli S, Totty NF, Hsuan JJ, Cour.
tneidge SA, Nature 1994 Apr 28; 368 (6474): 871-4).

Finally, another way to determine or map the binding domain (interaction mediating sequence) is to elucidate the fine structure of the complex using X-ray structural analysis, nuclear magnetic resonance or electron microscopy. It is to be.

Example 3 Study of the Interaction of a Protein from Archaeoglobus fulgidus Using the Yeast Two-Hybrid System (Y2H) The Gene Product Has an In Vitro Interaction Performing Sequence and / or Nucleic Acid Synthesis Activity Can be used as Archeoglobus fulgidus (Archaeoglobus fulgidus) gene coding region is amplified by PCR, cloned into the vector pGBT9 (column of Figure 9) and pGAD424 (row of Figure 9), yeast PJ69-4a (pGAD424). ) And PJ69-4α (against pGBT9) as a hybrid protein by gap repair. The positive control was also amplified by PCR and contained the vectors pGBT9 and pGAD42.
4 (see also row 1 of FIG. 9) in yeast strain PJ69-4a (see also
pGAD424) and PJ69-4α (for pGBT9) were expressed as hybrid proteins by gap repair. Diploid cells containing the two vectors were made by pairing. The expression of three independent reporters (HIS3, ADE2 and MEL1) was measured. Expression of the HIS3 and ADE2 genes leads to histidine or adenine prototrophy in this cell. Transcription of the MEL1 gene leads to the production of β-galactosidase enzyme whose activity can be detected by, for example, a color reaction. In the experiment shown in FIG.
Cells grown in histidine- and adenine-deficient media are cells that carry both vectors, and the expression products of these two vectors are linked together. The interaction of the fusion proteins leads to the reconstitution of a functional transcription factor that initiates transcription of the reporter gene. Production of the protein encoded on the reporter gene abolishes histidine and adenine auxotrophy. This is due to the fact that the binding of expression products initiates transcription, which allows the cells to grow.

All positive clones in this experiment were also positive for MEL1 gene expression. The two-hybrid system is operated by Clontech Company (yeast protocol handbook
, PT3024-1).

Example 4 Determination of Interaction Fulfillment Sequence of Af0497 In order to determine the interaction feasible sequence on the protein Af0497 (elongation protein) that causes the interaction with the gliding protein, first, the DNA of the gene of Af0497 was determined. Was obtained by defining an appropriate vector containing an extension protein containing the interaction-performing sequence. This DNA was then ultrasonically fragmented and ligated into the two vectors pGAD424 and pGBDU. This generated banks of various fragments of the gene in the two vectors. The yeast two-hybrid system was then used to determine which of these fragments could result in interaction with Af0335. For this purpose both banks were transformed in the appropriate yeast strains (pGAD424: PJ69-4a, pGBDU: PJ69-4α). Diploid cells containing the bank (Af0497) and gliding protein in a suitable vector for use in the two-hybrid system were obtained by pairing this yeast strain. All clones that activate the HIS2 reporter (see above) were isolated on histidine-free plates,
Inserts were selectively amplified by yeast colony PCR. The inserts were sequenced and their position on the Af0497 gene was determined. The results are shown schematically in Figure 8A: 1
All clones found, except for one, contained the carboxy terminus of an extension protein containing one or several interaction performance sequences. The smallest clone is 51
It was composed of amino acids.

In order to determine whether the homologous protein interaction-carrying sequence of another organism is also present at the carboxy terminus of that protein, Pyrococcus horikoshi (P
The experiment with the polymerase protein from S. yrococcus horikoshi) and the gliding protein was repeated. To this end, P. horikosii
A bank of large subunits of the polymerase from was prepared and tested for interaction with the gliding protein from P. horikoshii by the method described above. The interacting fragment also included the carboxy terminus of the extension protein.

To determine whether the interaction between the carboxy terminus of the protein Af0497 and the gliding protein is specific, the carboxy-terminal protein fragment of Af0497 was isolated from Archaeglobus fulgidus and other organisms. It was tested whether or not it could also interact with other proteins. No interaction could be measured, indicating that this binding is highly specific for gliding proteins.

Therefore, polymerase Taq did not bind to SCP (PCNA from Archaeoglobus fulgidus) and no interaction was measured in the yeast two-hybrid system. To test whether the carboxy terminus of the protein Af0497 results in an interaction with another protein fused to it, a fusion protein consisting of Taq and the 51 carboxy terminal amino acids of Af0497 was prepared. This protein was tested for interaction in the yeast two-hybrid system: fusion of the Af0497 interaction-competing sequence to Taq results in a specific interaction between Taq and the gliding protein Af0335, as shown in Figure 4. . The results of the corresponding Y2H experiment are shown in Figure 4.

This is due to the nature of this fragment (the carboxy terminus of the elongation protein Af0497) that leads to the interaction with the gliding protein Af0335, in fact the interaction with the gliding protein specific for Af0497. It is shown that it can be fused or transferred to another protein, especially another polymerase, to give rise to it.

Six proteins from A. fulgidus (see also FIGS. 5 and 5B) were used to measure interaction with PCNA. This indicates that all of these proteins contain similar interaction-carrying sequences that mediate their interaction with PCNA. That is, for example, polymerase δ large subunit (Af 0497, TREMBL number: 029753), polymerase δ small subunit (Af 1790, TREMBL number: 028484),
DP2 (Af 1722, TREMBL number: 028552), RPA2 (replication factor A), RFC2 (replication factor C) and
PCNA (Af0335, TREMBL number: 029912).

Such an interaction performance sequence is contained in the last 50 amino acids of protein Af0497. A survey of proteins that interact with PCNA showed that all contained a motif located immediately before the carboxy terminus of the amino acid sequence. FIG. 8B shows a list of related sequences, where conserved regions are indicated by black bars.

This motif is also conserved in other organisms and genes. FIG. 5 shows the results of searching for such interaction performance sequences and consensus sequences generated therefrom.

Example 5: Increasing the efficiency of PCR by using PCNA as SCP and the chimeric extension protein according to the invention This example shows the effect of PCNA on the efficiency of the PCR reaction. In this PCR reaction,
The 463 bp fragment was amplified from plasmid DNA. As shown in Figure 6A, Taq fusion protein was used as the polymerase. Reaction conditions for PCR were as follows: 0.4 mM of each dNTP (pH 8.3) and 20 pmol of each primer in one reaction. Array
The first primer with 5'-AGGGCGTGGTGCGGAGGGCGGT-3 '(SEQ ID NO: 13) and the second primer with the sequence 5'-TCGAGCGGCCGCCCGGGCAGGT-3' (SEQ ID NO: 14) were used.

It was found that the fusion of the 50 amino acid domain to the C-terminus of the native Taq DNA polymerase does not adversely affect the properties of this polymerase, as the Taq fusion protein itself already produces the product in the PCR reaction. So using this domain,
It can mediate the interaction of the sliding glaze protein PCNA from the Archaeoglobus fulgidus with the Taq fusion protein and increase the efficiency of the PCR reaction due to its nature as a processivity factor (this PC
This is reflected in the rather high yield of R product). The result is shown in FIG.

Example 6 Elongation Protein Af0497 of Archaeoglobus fulgidus and Archaeoglobus fulgidus
Yeast two-hybrid system for detecting the interaction between the gliding proteins of Af0335

FIG. 9 shows the results of a Y2H experiment, in which row A contains cells with empty pGAD424 vector (Clontech, Palo Alto, USA) such that the transcriptional activation domain is expressed. , Column B contains cells harboring the pGAD424 vector from which the Sacharomyces cerevesiae gene CDC48 is expressed as a fusion protein having the transcriptional activation domain; column C from which fusions having the transcriptional activation domain are contained. Archaeoglobus fulgidus (Archaeoglobus) as protein
cells containing the pGAD424 vector in which the gliding gene from fulgidus) is expressed; row D is cell-free, and row E is the fusion protein with the transcriptional activation domain from which Archeoglobus fulgidus (Archaeoglobus) is present. fulgi
cells containing the pGAD424 vector in which the elongation protein gene derived from dus) is expressed.

Column 1 contains cells with the empty pGBT9 vector (Clontech, Palo Alto, USA), column 2 from which the Saccharomyces cerevisiae gene UFD3 is expressed as a fusion protein with a DNA binding domain. Tandem 3 contains cells carrying the pGBT9 vector; tandem 3 contains cells carrying the pGBT9 vector from which the gliding protein from Archaeoglobus fulgidus is expressed as a fusion protein having a DNA binding domain; 4 is a fusion protein having a DNA-binding domain.
Includes cells carrying the pGBT9 vector in which the coupling protein from Archaeoglobus fulgidus is expressed; column 5 from which the fusion protein with the DNA-binding domain is Archeoglobus fulgidus (Archaeoglobus fulgidus).
gidus) -derived elongation protein is expressed in the cells containing the pGBT9 vector.

Example 7: Polymerase Chain Reaction of Genomic DNA with Chimeric Extension Proteins Example 7 describes the effect of sliding glaze proteins on the efficiency of PCR reactions on long DNA fragments using recombinant chimeric proteins of the invention. Show the impact. This PCR
In the reaction, a 4954 bp fragment was amplified from human genomic DNA. The recombinant chimeric protein according to the present invention was used as a polymerase. Conditions correspond to standard PCR reaction conditions: pH 8.3, 0.4 mM of each pNTP and 20 pmol of each primer in one reaction. The first primer (SEQ ID NO: 15): (5-AGGAACAACATATGACGCACTCT-3) and the second primer (SEQ ID NO: 16): (5'-TAGGTGGCCTGCAGTAATGTTAG-3 ') were used.

Only the recombinant chimeric protein of the invention having the sequence shown in FIG. 6A produces a clear and specific PCR product under the stimulation of the gliding protein PCNA, while the native Taq DNA polymerase. The same reaction mixture containing the did not produce the indicated product. This can be explained by the fact that the interaction between the gliding protein and the recombinant chimeric protein according to the invention leads to a lower dissociation of the recombinant chimeric protein according to the invention from the target DNA.

The drawings, which have already been mentioned in part in the examples, are explained in more detail below. All sequence alignments disclosed in the drawings below are Altschul, SF, Gish, W. Miller, W.
BLAS in accordance with J. Mol. Biol. 215, 403-410 (1990), Myers, EW and Lipman, DJ.
It was determined using the T algorithm.

FIG. 1: FIG. 1 shows that humans, Archaeglobus fulgidus, Methanococcus thermoautotrophicusm, PHBT (Pyrococcus ho) from various organisms, ie, elongation protein 1.
rikoshii) and Methanococcus janashii),
Sequence alignments of a total of 4 different extended protein domains are shown. In this figure, there is often a consensus sequence just below the juxtaposition, where variable positions are indicated by a single symbol. Amino acids in square brackets represent amino acids expressed in the one-letter code that may occur at special positions.

Amino acids are named according to standard IUPAC one-letter nomenclature and listed according to the prosite patterning standard. The following amino acids are often grouped together: G, A, V, L, I, M, P, F or W (amino acids with non-polar side chains), often also just under the sequence "$". S, T, N, Q, Y or C (amino acid having an uncharged polar side chain), K, R, H, D or E (amino acid having a charged and polar side chain), often as "&" immediately under the sequence Enter. Also, in the sequences or sequence protocols, X represents any amino acid or insertion or deletion.

FIG. 1 shows four different consensus sequences for different regions of the extension protein, where for example extension protein 3 is often found in eubacteria and extension protein 4 also contains Taq polymerase. , Often found in Pol type I polymerases. Thus, elongation protein 3 exhibits juxtaposition with the conserved region of the elongation protein from eubacteria and the consensus sequence derived therefrom.
The following genes are shown: DP3A_ECOLI: DNA Pol III, α subunit, Escherichia coli (Esch
erichia coli), BB0579: DNA Pol III, α subunit, Borrelia burgdorferi, DP3A_HELPY: DNA Pol III, α subunit,
Helicobacter pylori AA50: Aquifex aeolicus, section 50 and DP3A_SALTY: DNA Pol III, α subunit, Salmonella typhimurium).

FIG. 2: FIG. 2 shows a schematic representation of a possible form of the recombinant chimeric protein according to the invention, in which the sliding glaze, a factor that increases the synthetic activity of the polymerase, interacts. It is bound to an extension protein having nucleic acid synthesis activity via a domain containing an execution sequence.

FIG. 3: FIG. 3 shows a consensus array of four sliding glare. The following genes are shown as gliding region 1 : human PCNA and Archaeoglobus fulgidus, Methanococcus janashii, Pyrococcus horikoschi.
i) and Methanococcus themoauto
its ortholog from throphicus).

Region 2 represents the alignment of the second conserved region of sliding glazes from eukaryotes and Archae with consensus sequences derived therefrom. This juxtaposition is region 1
Established with the second area of sliding glaze already used above for.

Region 3 shows the conserved region of gliding glazes from eubacteria and the alignment of consensus sequences derived from them. The following genes are shown: AAPOL3B, DP3B_ECOLI, S
.TYPHIM, DP3B_PROMI, DP3B_PSEPU and DP3B_STRCO (AAPOL3B: Aquifex Aeolic
us Section 93: DP3B_ECOLI: DNA Pol III, β chain, Escherichia coli, S.TYPHI
M: DNA Pol III, β chain, Salmonella typhimurium, P3B_PROMI: DNA Pol III, β
Chain, Proteus mirabilis DP3B_PSEPU: DNA Pol III, β chain, Pseudomonas putida
DP3B_STRCO: DNA Pol III, β chain, Streptomyces coelicolor).

Region 4 shows a second conserved region of gliding glazes from eubacteria (see region 3 organisms) and an alignment of consensus sequences derived therefrom.

Figure 4 Figure 4 shows the interaction of chimeric proteins in the yeast two-hybrid system.
The results shown in Figure 4 demonstrate that the property of promoting interaction with the gliding protein Af0335 can be transferred to another protein, especially another polymerase, where it results in interaction with the gliding protein. is doing. SCP binds to itself, and SCP binds to Archaeoglobus fulgid.
It is shown to bind to a Taq fusion protein containing an interaction performance sequence derived from us).

FIG. 5: FIG. 5 shows three alignments of conserved regions of interaction-competing sequences from different organisms and to different genes: Sequence 1 (polymerase gene, Af0497 homolog): Archeoglobs.・ Archaeoglobus fulgidus, Pyrodictium ocultum
ccultum), Aeropyrum pernix, Pyrococcus glycovorans, Pyrococcus furiosus
riosus), Thermococcus gorgonarius, Pyrococcus abyssi, Pyrococcus
horikoshii), Thermococcus litoralis, Thermococcus fumicolans, and Methanococcus jannaschii. Sequence 2 (polymerase gene, Af1722 homologue): Archaeoglobus fulgidus, Methanococcus jan
naschii), Pyrococcus furiosus, Methanobacterium thermoautotrophicum, Pyrococcus horikoshii and Pyrococcus abysii.
Pyrococcus abyssi) and sequence 3 (Af, homologue of 1347), Archeoglobus fulgidus (Archaeoglobus)
fulgidus), Pyrococcus abyssi, Pyrococcus horikoshii, Methanobacterium thermoautoficum (Me
thanobacterium thermoautotrophicum) and Aeropyru pernix (Aeropyru
m pernix). The interaction performance sequence can be introduced into the chimeric protein of the present invention to bind SCP.

FIG. 5B: FIG. 5B shows two juxtapositions of conserved regions of interaction promoting sequences from different organisms and to different genes: Sequence 4 (RNase gene, Af 0621 homolog): Archaeoglobus frugidus
(Archaeoglobus fulgidus), Pyrococcus abyssi, Pyrococcus horikoshii and Arabidopsis thaliana and sequence 5 (polymerase gene): Archeoglobus fulgidus
bus fulgidus), Pyrococcus abyssi, Methanococcus thermoautotrophicus, Methanococcus janashii, Mus musculus
And Homo sapiens. The interaction performance sequence can be introduced into the chimeric protein of the present invention to bind SCP.

FIG. 5C: FIG. 5C shows a list of letters used in the classification of one or several different amino acids, where the nature of the amino acid or group thereof is listed under “class” and the symbols used. Are listed under "key" and the amino acids within that group are listed under "residues".

FIG. 6A: FIG. 6A shows the complete sequence of a preferred embodiment of the recombinant chimeric protein according to the present invention. In this case, the elongation protein, and thus the nucleic acid synthesis activity, was derived from Thermus aquaticus and the interaction performance sequence was derived from Archaeoglobus fulgidus.

FIG. 6B: FIG. 6B shows a general diagram illustrating the structure of a preferred embodiment of the recombinant chimeric protein according to the present invention. In this case, the elongation protein is Thermus acadisc (Thermus
aquaticus), and the interaction performance sequence is Archeoglobus fulgidus (
Archaeoglobus fulgidus).

FIG. 7: FIG. 7 shows the use of the recombinant chimeric protein according to the invention in PCR; its sequence is shown in FIG. 6A. In all columns of this 1% agarose gel, one band with a 463 bp size corresponding to the amplificate of the PCR reaction starting with plasmid DNA was observed (
PCR2.1 vector containing 463 bp fragment; Invitrogen, 9704 CH Groningen, The Netherlands), which was carried out under the following conditions: (95 ° C 5'denaturation; 30x {95 ° C 30 ''denaturation; 55 ° C 30 '' hybridization). 72 ° C. 40 ″ extension}; 72 ° C. and an extension of 7). 20 μL of 50 μL of reaction mixture was applied to the gel. The seventh column shows the size standard.

In the second row (PCNA 0.3 μg) and the third row (PCNA 2.4 μg) increasing amounts of gliding glaze protein (PCNA) was added as stimulant at a constant Mg ²⁺ ion concentration (2.5 mM). later,
It can be observed that the band intensity is increased compared to the first column (PCR reaction without PCNA). A further increase in the intensity of these bands, and thus an increase in the yield in the PCR reaction, was observed in columns 4 (no PCNA) and 5 (PCNA 0.3 μg) with increased PCNA at higher Mg ²⁺ ion concentrations (3.5 mM). And can be achieved with column 6 (PCNA 2.4 μg).

Figure 8A: Schematic of the entire fragment of Af0497 (see above) interacting with the gliding glaze protein (Af0335) from Archaeoglobus fulgidus. The arrow labeled "B" indicates a clone found as a fusion with the DNA binding domain and the arrow labeled "A" indicates a clone found as a fusion with the activation domain. .

FIG. 8B: FIG. 8B shows the following genes Af 0497-grUE, af 1195 Rfc, Af 0264 Rad2-Fen1, Af 0.
621 RNase H, C for Archaeoglobus fulgidus
The alignment of the terminal sequences is shown.

FIG. 9: FIG. 9 shows the results of the yeast two-hybrid test, yeast proteins ufd3 and cdc.
48 (2B; positive control), elongation protein and gliding protein (3E; and 5C in the opposite direction), gliding protein and gliding protein (3C) and coupling protein and gliding protein (4C) Proves the bond between. Fusion protein is vector pGBT9 (vector) in column 1 and pGBT in 2
T9 :: ufd3 (positive control), pGBT9 :: PCNA for 3, pGBT9 :: klUE for 4, pGBT9 :: grUE for 5
, Row A pGAD424 (vector), B pGAD424 :: cdc48 (positive control), C pGAD42
4 :: PCNA, empty in D, and pGAD :: grUE in E.

The interaction of proteins from Archaeoglobus fulgidus was demonstrated in the Y2H system. The gene product can be used in vitro Archeoglobus fulgidus (Archaeoglobus fulgidus) from the gene coding region is amplified by PCR, cloned into the vector pGBT9 (column of Figure 27) and pGAD424 (row of Figure 9), Yeast PJ69-4a (for pGAD424) and PJ69-4α (for pGBT9)
In), it was expressed as a hybrid protein by gap repair. The positive control was also amplified by PCR and contained vectors pGBT9 (see also tandem in Figure 9) and pGA.
D424 (see also row in Figure 9) and cloned into yeast strain PJ69-4a (pGAD424
) And PJ69-4α (for pGBT9) as a hybrid protein by gap repair. Diploid cells containing the two vectors were generated by pairing according to the grid shown in FIG. Expression of three independent reporters (HIS3, ADE2 and MEL1) was measured. Figure 9 shows growth on media lacking histidine and adenine. The cells grown in this experiment carry both vectors, and in addition the expression products of the two vectors bind to each other. Binding of this expression product initiates transcription, which leads to the elimination of histidine and adenine auxotrophy.

All positive clones in this experiment are also positive for MEL1 gene expression. The operation of this two-hybrid system is based on the Clontech Company (yeast protocol handbo
OK, TP3024-1).

Figure 10: PCR in all columns of this 1% agarose gel except column 5 showing size standards
20 μL of a 50 μL preparation of the amplification product of the reaction was applied to the gel under the following conditions: (95 ° C. 5 ′ denaturation; 35 × {95 ° C. 30 ″ denaturation; 47 ° C. 30 ″ hybridization; 7
PCR was performed on human genomic DNA with a size of 4954 base pairs under 2 ° C. 12 ′ extension}; 72 ° C. and an additional 10 extension (Roche Diagnostics, D-68305 Mannheim). 3rd row
(PCNA 2.4 μg), an increasing amount of gliding protein (Archaeoglobus fu
lgidus-derived PCNA) was added as a stimulant to the recombinant chimeric protein complex according to the present invention, and then compared to the first column (PCR reaction without PCNA) and the second column (PCNA 0.3 μg) at a constant level. One band was obtained at the Mg ²⁺ ion concentration (6 mM), while the same Mg ²⁺
With Taq DNA polymerase at ionic concentrations, no defined product could be produced in this PCR reaction (column 4).

The features of the invention disclosed in the description and the claims can be important individually as well as in any combination for implementing the invention in various ways.

[Sequence list]

[Brief description of drawings]

[Figure 1]   Figure 1 shows four sequence alignments of different extended protein domains.

[Fig. 2]   FIG. 2 shows a schematic representation of the recombinant chimeric protein according to the present invention.

[Figure 3]   FIG. 3 shows four consensus sequences of gliding proteins.

[Figure 4]   FIG. 4 shows a table of results for the yeast two-hybrid system.

[Figure 5]   Figure 5 shows the alignment of several conserved regions of the sequences responsible for the interaction.

FIG. 5B   FIG. 5B shows the alignment of some conserved regions of the sequences responsible for the interaction.

FIG. 5C   Figure 5C shows the alignment of some conserved regions of the sequences responsible for the interaction.

FIG. 6A shows the full sequence of one embodiment of the recombinant chimeric protein according to the present invention.

FIG. 6B   FIG. 6B shows a schematic of the basic structure of the chimeric protein according to the present invention.

FIG. 7 shows the results of polymerase chain reaction performed using the chimeric protein according to the present invention.

FIG. 8A shows Archaeoglobus fulgidus (Af033).
5 shows all fragments of Af0497 that can interact with the gliding protein of 5).

FIG. 8B shows an alignment of the C-terminal sequences of various genes from Archaeglobus fulgidus.

FIG. 9 shows the results of a yeast two-hybrid experiment showing the interaction of stretch proteins with gliding proteins. And

FIG. 10 shows the results of amplification of genomic DNA using the recombinant chimeric protein according to the present invention.

[Procedure for Amendment] Submission for translation of Article 34 Amendment of Patent Cooperation Treaty

[Submission date] October 29, 2001 (2001.10.29)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Claims

[Correction method] Change

[Contents of correction]

[Claims]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 5/10 C12Q 1/42 4H045 9/10 1/48 Z C12Q 1/42 1/68 A 1/48 G01N 33/58 A 1/68 C12N 15/00 ZNAA G01N 33/58 5/00 A (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, L, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB , GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA , UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Laser, Efa Germany, De-69121 Heidelberg, Fritz-Frei-Strasse 5 / Ar 37 (72) Inventor Kegur, Mann Freight Day 69214, Federal Republic of Germany Bae Ruhaimu, Haupt Strasse 131/4 F term (Reference) 2G045 AA35 DA13 FA16 FB02 4B024 AA11 AA20 BA10 CA04 CA07 GA11 HA01 HA15 4B050 CC01 CC05 DD02 LL03 LL05 4B063 QA01 QA18 QQ28 QQ33 QQ34 QQ42 QQ52 QR55 QR62 QS25 QS34 4B065 AA01X AA01Y AB01 AC14 CA29 CA46 4H045 AA11 BA41 CA11 DA76 DA89 EA50 FA74

Claims (44)

[Claims] 1. An interaction-mediating sequence results in the formation of a complex between a nucleic acid synthesis activity and a gliding protein, the complex being the nucleic acid synthesis activity and / or the gliding protein of which they are their natural interacting partners. A recombinant chimeric protein comprising: a) a first domain having nucleic acid synthesis activity; and b) an interaction mediating sequence, which is different from the complex formed with. 2. A moiety having nucleic acid synthesizing activity, wherein the moiety is derived from a basic protein comprising a moiety having nucleic acid synthesizing activity and an interaction mediating moiety, and at least one interaction mediating moiety, wherein: The interaction-mediating portion is different from the interaction-mediating portion of the basic protein. 3. The chimeric protein according to claim 2, wherein the protein contains an interaction-mediating portion of a basic protein. 4. A chimera comprising a portion having nucleic acid synthesis activity, wherein the portion is derived from a basic protein containing a portion having nucleic acid synthesis activity but no interaction mediation portion, and an interaction mediation portion. protein. 5. The interaction-mediating moiety mediates a binding between a nucleic acid synthesizing activity and a factor affecting the rate of synthesis of the nucleic acid synthesizing activity.
The chimeric protein according to any one of 1. 6. The chimeric protein according to claim 5, wherein the factor is a gliding protein. 7. The method according to any one of claims 1 to 6, characterized in that the nucleic acid synthesis activity comprises a consensus peptide sequence, wherein this sequence is selected from the group comprising SEQ ID NOs: 1, 2 and 3. The recombinant chimeric protein according to the item. 8. The method of claim 1-7, wherein the gliding protein comprises a consensus peptide sequence, wherein the sequence is selected from the group comprising SEQ ID NOs: 4, 5, 6 and 7. The recombinant chimeric protein according to any one of items. 9. The interaction mediating sequence comprises a consensus peptide sequence selected from the group comprising SEQ ID NOs: 8, 9, 10, 11 and 12, wherein any one of claims 1 to 8 is characterized. The recombinant chimeric protein according to 1. 10. Recombinant chimeric protein according to claim 9, characterized in that the interaction mediating sequence comprises the consensus peptide sequence of SEQ ID NO: 8. 11. The recombinant chimeric protein according to any one of claims 1 to 6, characterized in that the interaction mediating sequence is at the C-terminus of the sequence having nucleic acid synthesis activity. 12. Recombinant chimeric protein according to any one of claims 1 to 11, characterized in that the linker is located between the interaction mediating sequence and the sequence having nucleic acid activity. 13. Recombinant chimeric protein according to any one of claims 1 to 12, characterized in that the chimeric protein is thermostable. 14. The recombinant chimeric protein according to claim 1, wherein the protein has a DNA polymerase activity. 15. Recombinant chimeric protein according to claim 14, characterized in that said protein has a 3'-5 'exonuclease activity. 16. The recombinant chimeric protein according to any one of claims 1 to 15, wherein the protein has RNA polymerase activity. 17. The recombinant chimeric protein according to claim 1, wherein the protein has a reverse transcriptase activity. 18. Recombinant chimeric protein according to claim 14, characterized in that the uptake rates of dNTP and ddNTP due to nucleic acid synthesis activity differ by a factor of less than 5. 19. A complex comprising: a) the recombinant chimeric protein according to any one of claims 1 to 18; and b) a gliding glaze protein. 20. The complex of claim 19, wherein the gliding protein comprises a consensus peptide sequence selected from the group comprising the sequences of SEQ ID NOs: 4, 5, 6 and 7. 21. The complex according to claim 19 or 20, further comprising a nucleic acid. 22. A nucleic acid encoding the recombinant chimeric protein according to any one of claims 1-18. 23. A vector comprising the nucleic acid of claim 22. 24. The vector of claim 23, which is an expression vector. 25. A cell containing the vector according to claim 23 or 24. 26. Use of the recombinant chimeric protein according to any one of claims 1 to 18 for extending a nucleic acid. 27. Use of the recombinant chimeric protein according to any one of claims 1-18 for amplifying nucleic acids. 28. Any one of claims 1-18 for reverse transcription of RNA into DNA.
Use of the recombinant chimeric protein according to section. 29. Use of a recombinant chimeric protein according to any one of claims 1-18 for sequencing DNA. 30. In one or several separate containers: a) the recombinant chimeric protein according to any one of claims 1-18, and / or b) any one of claims 19-21. And preferably c) optionally containing at least one primer, buffer, nucleotides, cofactor, and / or pyrophosphatase, extending and / or amplifying and / or reversing nucleic acid. Reagent kit for copying and / or sequencing and / or labeling. 31. Kit according to claim 30, characterized in that it comprises deoxynucleotides and / or their derivatives in addition to substances a) and / or b) for amplifying nucleic acids. 32. The kit according to claim 30, which comprises a DNA polymerase having 3′-5 ′ exonuclease activity. 33. The method according to claim 30, characterized in that it comprises substances a) and / or b) having reverse transcriptase activity, and preferably deoxynucleotides and / or derivatives thereof for reverse transcription. Kit. 34. Kit according to claim 30, characterized in that it comprises dideoxynucleotides or / and derivatives thereof for sequencing in addition to deoxynucleotides or ribonucleotides or / and derivatives thereof. 35. At least one primer, under hybridization conditions, to the nucleic acid to be extended or at least one strand thereof, wherein the primer is sufficiently complementary to a part or flanking region of the nucleic acid to be extended. And the primer is extended by a polymerase in the presence of nucleotides, the method according to any one of claims 1 to 18 for template dependent extension of a nucleic acid. A method characterized in that the modified chimeric protein is used as a polymerase and, preferably, a gliding protein is present during the reaction. 36. At least two primers to the nucleic acid to be amplified under hybridization conditions, each of the two primers being complementary to a portion or flanking region of the nucleic acid to be amplified, And the primer is extended by a polymerase in the presence of nucleotides, the method for amplifying a nucleic acid comprising using the chimeric protein according to any one of claims 1 to 18 as a polymerase. And, preferably, adding gliding protein to the reaction. 37. Performing a polymerase chain reaction, 37. The method according to claim 36, characterized in that 38. The reaction mixture comprises two DNA polymerases, at least one of which has 3'-5 'exonuclease activity, and which 3'-5' exonuclease activity is due to the recombinant chimeric protein or to additional 38. The method of claim 37, characterized in that it is added to the reaction mixture by a polymerase. 39. Two recombinant chimeric proteins according to any one of claims 1 to 18 are present in the reaction, one is the protein according to claim 14 and the other is according to claim 15. 39. The method according to claim 38, characterized in that it is a protein of 40. Template-dependent according to Sanger's method using deoxynucleotides and dideoxynucleotides or their derivatives to sequence nucleic acids starting from primers complementary to the region flanking the nucleic acid to be sequenced. 36. The method according to claim 35, characterized in that sex extension or reverse transcription is carried out. 41. Introducing a label during nucleic acid extension, 36. The method of claim 35, characterized in that 42. Use of a reagent selected from the group comprising a labeled primer, a labeled deoxynucleotide and its derivative, a labeled dideoxynucleotide and its derivative, and a labeled ribonucleotide and its derivative. 42. The method of claim 41, wherein: 43. A method for labeling a nucleic acid by creating a single break in the phosphodiester bond of a nucleic acid chain and replacing the nucleotide at this break with a labeled nucleotide with the aid of a polymerase. A recombinant chimeric protein according to any one of claims 1 to 18 is used as a polymerase. 44. An antibody against the chimeric protein according to any one of claims 1-18.