CA2379165A1 - Chimeric proteins - Google Patents

Abstract

The invention relates to a recombinant chimeric protein comprising a) a first domain with a nucleic acid synthesis activity and b) an interaction mediating sequence, whereby said interaction mediating sequence can form a complex through the nucleic acid synthesis activity and a slide bracketing protein.
Said complex is different from the complex formed through the nucleic acid synthesis activity and/or the slide tying protein with their natural interaction partner(s).

Description

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Chimeric proteins The present invention concerns recombinant chimeric proteins which have a nucleic acid synthesis activity, complexes containing these proteins, nucleic acids encoding these proteins, vectors and cells containing these proteins, an antibody directed against them, applications of these proteins, kits containing them as well as methods for the elongation, amplification, reverse transcription, sequencing and labelling of nucleic acids.
Many proteins are not present in the cell as monomers but as part of a functional multimeric complex. Examples of such complexes have been described in almost all fields of cell biology (e.g. transcription, translation, replication, cytoskeleton, signal transduction, mRNA processing).
The interaction or binding of a protein with or to another protein which is referred to in the following as the donor protein or acceptor protein is effected by certain amino acids or amino acid sequences which, in the folded protein, are usually on the surface of the protein and are responsible for the specific binding or interaction to or with the partner. These amino acids or such an amino acid sequence is referred to herein in the following as the "interaction-effecting sequence" or interaction-mediating sequence. Persons skilled in the art know that the amino acids involved in the formation of an interaction are not necessarily directly adjacent to one another in the primary amino acid sequence but rather that positions within the amino acid sequence of the protein or peptide that are conserved to a greater or lesser degree are responsible for the interaction. If reference is made in the following to the determination of a sequence that mediates an interaction or effects an interaction, this is intended to mean both aforementioned aspects. In this connection donor proteins are understood as those proteins which interact with another protein and optionally bind another protein. Likewise acceptor proteins are understood as those proteins which interact with another protein and may bind another protein.
There are a number of methods for determining the interaction-effecting sequence of a protein-protein interaction site. These include (i) the determination of the three-dimensional structure of a complex of donor and/or acceptor protein by X-ray structural analysis or (ii) NMR methods. Another method is (iii) to reconstitute a complex binding in vitro using recombinant proteins and to determine the sequences) which effect the interaction by specifically changing the donor or acceptor protein. Such specific changes include the mutation of individual amino acids, for example to form alanine (alanine scanning) or the deletion of sequences in the protein. If the change or deletion of an amino acid or sequence leads to the loss of the interaction or binding activity then it is a part of the sequence effecting the interaction and conversely if the change or deletion of an amino acid or sequence does not lead to the loss of the interaction or binding activity then it is not part of the interaction-effecting sequence. The interaction-effecting sequence can be defined in this manner.
Another method for determining the interaction-effecting sequence of a protein is based on the use of a two-hybrid system also abbreviated in the following as "Y2H". Y2H systems are based on the expression of a protein having a detectable activity (such as the enzyme dihydrofolate reductase) as two non-covalently linked parts. This protein is inactive when the two parts are not in spatial vicinity to one another. The two proteins to be examined which are known to interact with one another and thus can form a complex i.e. donor protein and acceptor protein, or which are to be investigated in this regard, are each fused with one of the two parts of the protein having the detectable activity (such as dihydrofolate reductase) to form a fusion protein and expressed, which results in the formation of two fusion proteins. If the fused donor protein binds the fused acceptor protein, the two halves of the detectable protein (such as dihydrofolate reductase) come into spatial proximity. This restores the activity of the protein which can then be detected.
Suitable proteins having detectable activity are enzymes (dihydrofolate reductase, beta-galactosidase), signal transduction proteins (Cdc25 from Saccharomyces cerevisiae) or transcription activators (Gal4, LexA-VP16). The determination of the binding region using the two hybrid system is based on the same considerations as with the in vitro reconstitution of binding: If the change or deletion of an amino acid or sequence leads to the loss of binding activity, then it is part of the interaction-effecting sequence and conversely if the change or deletion of a sequence does not lead to the loss of binding activity then it is not part of the interaction-effecting sequence.

. .. _3_ In addition the interaction-effecting sequence can be defined by examining the interaction of numerous fragments of the protein and determining which parts of the protein are always present in the interacting fragments. This region which is always present is the interaction-effecting sequence. In order to identify the amino acids in an interaction-effecting sequence that are essential for the interaction or binding, it is possible to change individual amino acids by targeted mutations. A loss or increase in the binding activity indicates that these positions are directly involved in the interaction.
The complexes that are particularly preferred for in vitro applications include the thermostable complexes of prokaryotic and eukaryotic replication apparatuses which often contain polymerases as an important enzymatic activity.
As already mentioned the interaction between proteins in vivo plays a major role for example in the replication of nucleic acids in biological systems. Thus highly processive replication mechanisms are known which are, on the one hand, cellular mechanisms and, on the other hand, the replication mechanisms which occur in the bacteriophages T4 and T7.
The replication apparatus comprises many components. These include among others a) proteins having polymerase activity, b) proteins which are involved in the formation of a clamp structure, the function of the clamp structure being, among others, to bind a polymerase activity to its template and to stabilize the binding and thus to change the dissociation constant of the complex of polymerase and nucleic acid accordingly, c) proteins which load the clamp onto the template, d) proteins which stabilize the template and optionally e) proteins which guide the polymerase onto the template.
Proteins having polymerase activity are understood herein in particular as those proteins which are able to bind one or several nucleotides or nucleosides to a nucleotide or nucleoside or polynucleotide or polynucleoside. In each of the above cases these can be ribonucleotides/ribonucleosides or deoxynucleotides/deoxy-nucleosides or polymers thereof. Hence these proteins include, apart from DNA
polymerases, also RNA polymerases irrespective of whether a template is required or not required for the polymerization reaction of the protein. These proteins having polymerase activity are thus also proteins having nucleic acid synthesis activity.
These also include the elongation proteins known as such in the prior art. An elongation 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:
Uses RNA as a template, uses DNA as a template, synthesizes RNA, synthesizes DNA, exonuclease activity in the 5'-3' direction or exonuclease activity in the 3'-5' direction, strand displacement activity and processivity or non-processivity.
DNA polymerases belong to a group of enzymes which use single-stranded DNA as a template for the synthesis of a complementary DNA strand. These enzymes play a major role in nucleic acid metabolism including the processes of DNA
replication, repair and recombination. DNA polymerases have been identified in all cellular organisms from bacterial to human cells, in many viruses as well as in bacteriophages (Kornberg, A. & Baker, T.A. (1991) DNA Replication WH
Freeman, New York, NY). The archaebacteria and eubacteria are usually combined to form the prokaryote group which are organisms without a real cell nucleus in contrast to the eukaryotes which are organisms with a real cell nucleus. A
common feature of many polymerases from the diverse organisms is often a similarity of the amino acid sequence and a similarity of structure (Wang, J., Sattar, A.K.M.A.;
Wang, C.C., Karam, J.D., Konigsberg, W.H. & Steitz, T.A. (1997) Crystal Structure of pol a family replication DNA polymerase from bacteriophage RB69.Ce11 89, 1087-1099). Organisms such as humans have numerous DNA-dependent polymerases which are, however, not all responsible for DNA replication but some also carry out DNA repair. Replicative DNA polymerases are usually composed in vivo of protein complexes with several units which replicate the chromosomes of the cellular organisms and viruses. A general property of these replicating polymerases is in general a high processivity which means their ability to polymerise thousands of nucleotides without dissociating from the DNA template (Kornberg, A. & Baker, T.A. ( 1991 ) DNA Replication, WH Freeman, New York, NY).
DNA polymerases are characterized, among others, by two properties, their elongation rate i.e. the number of nucleotides which they can incorporate per second into a growing DNA strand and their dissociation constant. If the polymerase _5_ dissociates again from the strand after each step of incorporating one nucleotide into the growing chain (i.e. one elongation step occurs per binding event), then the processivity has the value 1 and the polymerase is not processive. If the polymerase remains connected to the strand for repeated nucleic acid incorporations, then the elongation or replication modus and thus also the polymerase is referred to as processive and can reach a value of several thousand (see also: Methods in Enzymology Volume 262, DNA replication, edited by J.L. Campbell, Academic Press 1995, pp. 270-280).
The proteins mentioned under b) form structures which are either open or closed, for example circular or semi-circular structures. Such structures can be formed by one or several species of proteins. One of the said protein species may have a polymerase activity.
The proteins responsible for the formation of these structures are referred to in the following as "sliding clamp proteins" or "clamp proteins" provided they have no polymerase activity.
It is known that the replication apparatus in archaea is similar to the eukaryotic replication apparatus although the genome organisation in eukaryotes and archaea is completely different and the cellular structure of the eubacteria is similar to that of the archaea (Edgell, D.R. and Doolittle, W.F. (1997), Archaea and the origins) of DNA replication proteins, Cell 89, 995-998).
The sliding clamp is frequently bound to an elongation protein via one or several other proteins, in other words it is coupled to the elongation protein. Such a coupling protein is referred to herein in the following as a coupling protein whereby the coupling may take place via a plurality of coupling proteins.
The three-dimensional structure of various sliding clamp proteins has already been determined. The overall structure of these sliding clamps is very similar; the pictures of the circular total protein structure of PCNA, of the (3 subunit and gp45 rings are superimposable when laid on top of one another (Kelman, Z. &
O'Donnel, M. (1995) Structural and functional similarities of prokaryotic and eukaryotic ~- -6_ sliding clamps. Nucleic Acids Res. 23, 3613-3620). Each ring has comparable dimensions and a central opening which is large enough to encircle duplex DNA
i.e.
a DNA double strand composed of the two complementary DNA strands.
The sliding clamp cannot position itself in vivo around the DNA but must be clamped around the DNA. In prokaryotes and eukaryotes such a protein complex is composed of numerous subunits. The protein complex recognises the 3'-end of the primer in the "primer-template duplex" which is to be elongated to form a longer double strand by incorporating nucleotides and positions the sliding clamp around the DNA.
In the case of the bacteriophage T7 the same object i.e. a processive DNA
synthesis, as defined below, is achieved by means of a protein complex with a different structure. The phage expresses its own catalytic polymerase, T7 polymerase, the gene product of gene 5 which binds to a protein from the host Escherichia coli i.e.
thioredoxin and enables a highly processive DNA replication as a replicase. In this case there is also clamp formation but this clamp does not have the same structure as for example in the case of the eukaryotic PCNA.
It is often necessary, as for example in the case of human polymerase 8, for proteins (coupling proteins) to make the connection between the catalytically active part of the polymerase and a processivity factor. A processivity factor is a compound or a molecule which influences the processivity of a polymerase and preferably increases it. Sliding clamp proteins are examples of processivity factors. In humans this coupling protein is a small subunit of the 8 polymerase (Zhang, S.-J., Zeng, X.-R., Zhang, P., Toomey, N.L., Chuang, R.Y., Chang, L.-S., and Lee, M.Y.W.T. (1994).
A conserved region in the amino terminus of DNA polymerase 8 is involved in proliferating cell nuclear antigen binding, J. Biol. Chem. 270, 7988-7992).
However, in the case of T7 polymerase the processivity factor binds the catalytic unit of the polymerase directly without involvement of a~coupling protein.
In vitro applications of proteins having nucleic acid synthesis activity such as polymerases or elongation proteins are widespread in the prior art e.g. for the polymerase chain reaction (PCR), nucleic acid sequencing or reverse transcription.

- -7_ For most in vitro applications such as PCR or sequencing processes, processivity is a desired property that, however, the thermostable enzymes of the prior art that are used in these reactions only have to a slight extent.
The US patents 4,683,195, 4,800,195 and 4,683,202 describe the application of such thermostable DNA polymerases in the polymerase chain reaction (PCR). In PCR
DNA is newly synthesized using primers, templates (also referred to as matrices), nucleotides, a DNA polymerase, an appropriate buffer and suitable reaction conditions. A thermostable polymerase which survives the cyclic thermal melting of the DNA strands is preferably used for this PCR. Thus Taq DNA polymerase is often used (LJS patent 4,965,188). However, the processivity of Taq DNA
polymerase is relatively low compared to that of T7 polymerase.
DNA polymerases are also used for DNA sequence determination (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 the Taq polymerase mentioned above (US patent 5,075,216) or the polymerase from Thermotoga neapolitana (WO 96/10640) or other thermostable polymerases. Recent methods couple the exponential amplification and sequencing of a DNA fragment in one step so that it is possible to directly sequence genomic DNA. One of the methods, the so-called DEXAS method (Nucleic Acids Res. 1997 May 15:25(10):2032-2034 Direct DNA sequence determination from total genomic DNA. Kilger C, Paabo S, Biol Chem. 1997 Feb;
378(2):99-105 Direct exponential amplification and sequencing (DEXAS) of genomic DNA. Kilger C, Paabo S and DE 19653439.9 and DE 19653494.1 ), uses a polymerase with a reduced ability to discriminate against dideoxynucleotides (ddNTPs) compared to deoxynucleotides (dNTPs) as well as a reaction buffer, two primers which are preferably not present in equimolar amounts and the above-mentioned nucleotides in order to then obtain a complete, sequence-specific DNA
ladder of a fragment in several cycles which is flanked by the primers. A
further development of this method comprises the use of a polymerase mixture in which one of the two polymerases discriminates between ddNTPs and dNTPs whereas the second has a reduced discrimination ability (Nucleic Acids Res. 1997 May 1 S;
25(10):2032-2034 Direct DNA sequence determination from total genomic DNA.
Kilger C, Paabo S).

_8_ DNA polymerises are also used for the reverse transcription of RNA into DNA.
In this case RNA serves as a template and the polymerise synthesizes a complementary DNA strand. The thermostable DNA polymerise from the organism Thermus thermusphilis (Tth) (US patent 5,322,770) is for example used in this case.
Some polymerises may also have a "proof reading" activity i.e. a 3'-5' exonuclease activity. This property is particularly desirable when the product to be synthesized should be produced with a low rate of nucleotide incorporation errors. The polymerises from the organism Pyrococcus wosei are an example of this.
The majority of the elongation proteins mentioned above that are used in the aforementioned applications are not actually replication enzymes in vivo but are mostly enzymes which are assumed to be involved in DNA repair which is why their processivity is relatively small. In addition each organism has many polymerises that have numerous properties.
As mentioned above such elongation proteins have for example the following properties: Use of RNA as a template, use of DNA as a template, synthesis of RNA, synthesis of DNA, exonuclease activity in the 5'-3' direction and exonuclease activity in the 3'-5' direction, strand displacement activity, processivity or non-processivity or thermostability or thermosensitivity. However, in vivo it is frequently the case that a protein complex combines one or several of these properties. In particular replication complexes are often present in vivo whose processivity is increased, as described above, by the presence of a sliding clamp protein.
In addition to the aforementioned components of replication apparatuses, other DNA-modifying proteins are also often found in vivo in large complexes with other proteins. It is often the case in such complexes that a DNA-modifying activity such as terminal transcriptase activity from a first protein is combined with one or several activities introduced into the complex by an additional protein such as exonuclease activity. DNA-modifying activity is understood herein as any enzymatic activity which leads to a chemical, physical or structural change of an initial nucleic acid. It is also sometimes the case that a DNA-modifying activity only occurs when there is an interaction with at least one further protein. It is also possible that a DNA-modifying activity is reduced or increased by interaction with at least one further protein. Hence in vivo a protein complex is often formed which for example carries the sum of the individual activities or whose activity is improved compared to the individual activity.
The above-mentioned shows that in vivo complexes which contain a nucleic acid synthesis activity often have additional desirable properties for technical, i.e. in vitro, applications which go beyond the actual nucleic acid synthesis activity and are contributed by other components forming the complex.
A direct technical application of such in vivo complexes e.g. for DNA
sequencing, performing a polymerase chain reaction or introducing labels into nucleic acids has previously been unsuccessful for a number of reasons. One reason was and is the lack of knowledge of all factors or individual components that are involved in forming the complex of interest. Another reason is that the complex comprising several components also has one or several undesired properties in addition to the desired properties.
An alternative approach for the in vitro use of in vivo complexes is to combine components of different origins but having the desired property and in this manner to form the complex having the desired properties. Such a property can for example be a higher processivity. A practical hurdle to this approach was that the individual components forming the complex which would have to interact with one another did not do this or only very poorly and hence it was not possible to give the complex the desired properties.
Hence the object of the present invention is to provide proteins having a nucleic acid synthesis activity which have an increased processivity.
A further object of the present invention is to provide a method which allows the construction of such proteins.

' - l~-Finally it is an object of the invention to provide methods for the amplification, especially by PCR, and sequencing of nucleic acids which allow longer nucleic acids to be amplified and/or sequenced.
The object is achieved according to the invention by a recombinant chimeric protein comprising a) a first domain with nucleic acid synthesis activity and b) an interaction-mediating sequence whereby the interaction-mediating sequence results in the formation of a complex of nucleic acid synthesis activity and sliding clamp protein and the complex is different from the complex that the nucleic acid synthesis activity and/or the sliding clamp protein form with their natural interaction partner(s).
In a further aspect of the invention the object is achieved by a chimeric protein comprising a part having nucleic acid synthesis activity whereby this part is derived from a part having nucleic acid synthesis activity and a basic protein containing an interaction-mediating component, and at least one interaction-mediating component wherein the interaction-mediating component is different from the interaction-mediating component of the basic protein.
In one embodiment the protein contains the interaction-mediating component of the basic protein.
In a further aspect the object is achieved by a chimeric protein comprising a part having a nucleic acid synthesis activity, the part being derived from a basic protein which comprises a part having nucleic acid synthesis activity but no interaction-mediating part, and an interaction-mediating part.
In the chimeric proteins according to the invention provision can be made for the interaction-mediating part to mediate binding between the nucleic acid synthesis activity and a factor which influences the synthesis rate of the nucleic acid synthesis activity. In a preferred embodiment the factor is a sliding clamp protein.
In one embodiment the nucleic acid synthesis activity is intended to comprise a consensus peptide sequence, the sequence being selected from the group comprising the sequences SEQ ID NO.: l, 2 and 3.
In a further embodiment the sliding clamp protein can comprise a consensus peptide sequence, the sequence being selected from the group comprising the sequences SEQ >D NO.: 4, 5, 6 and 7.
In yet a further embodiment the interaction-mediating sequence can comprise a consensus peptide sequence which is selected from the group comprising the sequences SEQ >D NO.: 8, 9, 10, 11 and 12. In a particularly preferred embodiment the interaction-mediating sequence comprises a consensus peptide sequence according to SEQ ID NO.: 8.
In a further embodiment the interaction-mediating sequence is at the C-terminal end of the sequence carrying the nucleic acid synthesis activity.
In a further embodiment a linker is located between the interaction-mediating sequence and the sequence carrying the nucleic acid activity.
Furthermore in one embodiment of the chimeric proteins according to the invention the recombinant chimeric proteins can be thermostable.
In one embodiment of the chimeric proteins according to the invention the protein can have a DNA polymerase activity. It is particularly preferred that the proteins have a 3'-5' exonuclease activity.
In a further embodiment of the chimeric proteins according to the invention the protein has an RNA polymerase activity.

In a further embodiment of the chimeric proteins according to the invention the protein has a reverse transcriptase activity.
In yet a further preferred embodiment of the chimeric proteins according to the invention the incorporation rate of dNTPs and ddNTPs by the nucleic acid synthesis activity differs by a factor of less than 5.
In a further aspect of the invention the object is achieved by a complex comprising a) a recombinant chimeric protein according to the invention and b) a sliding clamp protein.
In a preferred embodiment of the complex according to the invention the sliding clamp protein contains a consensus peptide sequence which is selected from the group comprising the sequences according to SEQ >D NO.: 4, 5, 6 and 7.
In a further embodiment the complex additionally contains a nucleic acid.
In a further aspect the object is achieved by a nucleic acid which codes for a chimeric protein according to the invention and especially for a recombinant protein.
In yet a further aspect the object is achieved by a vector containing the nucleic acid according to the invention. In a preferred embodiment the vector is an expression vector.
In a further aspect the object is achieved by a cell which contains the vector according to the invention.
The object is also achieved by the use of the chimeric protein according to the invention to elongate nucleic acids.

. -13-The object is also achieved by the use of the chimeric protein according to the invention to amplify nucleic acids.
The object is also achieved by the use of the chimeric protein according to the invention for the reverse transcription of RNA into DNA.
The object is also achieved by the use of the chimeric protein according to the invention to sequence nucleic acids and in particular DNA.
In yet a further aspect the object is achieved by a kit and in particular a reagent kit for the elongation and/or amplification and/or reverse transcription and/or sequencing and/or labelling of nucleic acids which comprises in one or several containers:
a) a chimeric protein according to the invention which is preferably a recombinant protein and/or b) a complex according to the invention and c) preferably optionally at least one primer, buffer, nucleotides, cofactors and/or pyrophosphatase.
In a preferred embodiment of the kit it contains deoxynucleotides or/and derivatives thereof in addition to the substances a) and/or b) to amplify nucleic acids.
In a further embodiment the kit contains a DNA polymerase having 3'-5' exonuclease activity.
1n another embodiment the kit contains substances according to a) and/or b) which have reverse transcriptase activity and preferably deoxynucleotides and/or derivatives thereof for reverse transcription.
The kit according to the invention can contain dideoxynucleotides or/and derivatives thereof for sequencing in addition to deoxynucleotides or ribonucleotides or/and derivatives thereof.

In a further aspect the object is achieved by a method for the template-dependent elongation of nucleic acids in which the nucleic acid to be elongated or at least one strand thereof is provided with at least one primer under hybridization conditions whereby the primers are sufficiently complementary to a part of or to a flanking region of the nucleic acid to be elongated and the primer is elongated by a polymerase in the presence of nucleotides wherein a chimeric protein according to the invention is used as the polymerase and preferably a sliding clamp protein is present in the reaction.
In yet a further aspect the object is achieved by a method for the amplification of a nucleic acid in which 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 part of or to a flanking region of the nucleic acid to be amplified and the primers are elongated by a polymerase in the presence of nucleotides wherein a chimeric protein according to the invention and in particular a recombinant chimeric protein is used as the polymerase and a sliding clamp protein is preferably added to the reaction.
In a preferred embodiment a polymerase chain reaction is carned out.
In an especially preferred embodiment the reaction mixture contains two DNA
polymerases of which at least one has a 3'-5' exonuclease activity and the 3'-5' exonuclease activity is either added to the reaction mixture by way of the chimeric protein or by an additional polymerase.
In yet a further embodiment two chimeric proteins are present and in particular recombinant chimeric proteins wherein one of the proteins has a DNA polymerase activity and the other has a 3'-5' exonuclease activity.
In the method according to the invention for template-dependent elongation, nucleic acids can be sequenced in one embodiment starting with a primer which is complementary to a region neighbouring the nucleic acid to be sequenced, and a template-dependent elongation or reverse transcription is carried out using - IS -deoxynucleotides and dideoxynucleotides or derivatives thereof according to the method of Singer.
In a further embodiment of the method according to the invention for template-dependent elongation at least one label can be introduced during the elongation of the nucleic acids.
In a particularly preferred embodiment an agent is used which is selected from the group comprising labelled primers, labelled deoxynucleotides and derivatives thereof, labelled dideoxynucleotides and derivatives thereof and labelled ribonucleotides and derivatives thereof.
In yet a further aspect the object is achieved by a method for labelling nucleic acids by generating single breaks in phosphodiester bonds of the nucleic acid chain and replacing a nucleotide at the breakage points by a labelled nucleotide with the aid of a polymerise wherein a chimeric protein according to the invention and in particular a recombinant chimeric protein is used as the polymerise.
In yet a further aspect the object is achieved by a method for producing a chirneric protein which comprises a base sequence and a heterologous interaction-effecting sequence and binds to an interaction partner or such a binding is strengthened as a result of the interaction-effecting sequence, wherein a) an interaction system comprising a protein referred to as donor protein and a protein referred to as acceptor protein is used to determine which sequence of the donor protein or acceptor protein effects the interaction between the two interaction partners and b) the interaction-effecting sequence is introduced into a recipient protein that is different from the donor protein and acceptor protein and contains the base sequence.

In one embodiment the donor protein and acceptor protein form a complex which binds the nucleic acid.
In a further embodiment the donor protein and the acceptor protein form a complex which has an activity that is selected from the group comprising polymerase activity, DNA binding activity, RNA binding activity, 5'-3' exonuclease activity, 3'-5' exonuclease activity and ligase activity.
In a preferred embodiment the donor protein is selected from the group comprising elongation protein, sliding clamp proteins, sliding clamp loader protein and coupling proteins.
In a further embodiment the acceptor protein is selected from the group comprising elongation protein, sliding clamp proteins, sliding clamp loader protein and coupling proteins.
In yet a further embodiment the recipient protein is selected from the group comprising elongation protein, sliding clamp proteins, sliding clamp loader protein and coupling proteins.
In one embodiment of the method according to the invention step a) can be repeated several times and a consensus sequence can be determined from the interaction-effecting sequences determined in this manner which represents an interaction-effecting sequence and introduced in step b) as the interaction-effecting sequence into a recipient protein that is different from the donor protein and acceptor protein and contains a base sequence.
In a further aspect the object is achieved by a chimeric protein which is obtainable by the method according to the invention. In a preferred embodiment the base sequence is a part of the amino acid sequence of a protein that is selected from the group comprising elongation proteins, sliding clamp proteins, sliding clamp loader proteins and coupling proteins.

Finally the object is also achieved by an in vitro complex for the template-dependent elongation of nucleic acids comprising a sliding clamp protein and an elongation protein wherein at least one of the proteins is a chimeric protein according to the invention. In this connection an embodiment is particularly preferred in which the complex is thermostable.
In connection with the present invention any of the chimeric proteins according to the invention can be a recombinant chimeric protein.
The object is also achieved according to the invention by a recombinant chimeric protein comprising a) a first domain having nucleic acid synthesis activity and b) an interaction-mediating sequence characterized in that the interaction-mediating sequence results in the formation of a complex of nucleic acid synthesis activity and sliding clamp protein, the complex being different from the complex that the nucleic acid synthesis activity and/or the sliding clamp protein form with their natural interaction partner(s).
Hence the recombinant chimeric protein according to the invention enables a nucleic acid synthesis activity to be bound to a sliding clamp protein that cannot bind the nucleic acid synthesis activity as it for example occurs naturally.
Hence the natural interaction partner is a partner that can be present bound to the nucleic acid synthesis activity under normal physiological conditions within an organism.
The object is achieved according to the invention by a chimeric protein comprising a part having nucleic acid synthesis activity, the part being derived from a part having a nucleic acid synthesis activity and a basic protein containing an interaction-mediating part, and at least one interaction-mediating part the interaction-mediating part being different from the interaction-mediating part of the basic protein, and by a chimeric protein comprising a part having nucleic acid synthesis activity, the part being derived from a basic protein which comprises a part having nucleic acid synthesis activity but no interaction-mediating part and an interaction-mediating part.

According to the invention the object is additionally achieved by a method for the template-dependent elongation of nucleic acids wherein the nucleic acid to be elongated or at least a strand thereof is provided with at least one primer under hybridization conditions, the primer being sufficiently complementary to a part of the nucleic acid to be elongated or to a flanking region thereof and a primer elongation by a polymerase is carned out in the presence of nucleotides characterized in that a recombinant chimeric protein according to the invention is used as the polymerase and that a sliding clamp protein is preferably present in the reaction.
In the sense of the present invention "recombinant" is for example understood to mean that the chimeric protein is for example produced by genetic engineering (see "Gentechnologie, Rompp Basislexikon Chemie, Georg Thieme Verlag" 1998) or for example when it is chemically synthesized.
Other embodiments arise from the subclaims.
The basis of the invention is the surprising discovery that starting with a protein having nucleic acid synthesis activity referred to herein in the following as the basic protein, it is possible to increase the processivity of this protein or more exactly the nucleic acid synthesis activity by interacting it with a factor such as a sliding clamp protein that increases the processivity whereby the basic protein as such cannot interact with this factor or if it can interact with this factor, it is not associated with an increase in processivity. This is achieved by providing the protein carrying the nucleic acid synthesis activity with a group of several amino acids, typically in the form of a consecutive amino acid sequence, which is referred to herein as the interaction-mediating or interaction-effecting sequence, and only as a result of which is an interaction possible between the said protein and a factor increasing the processivity. This overcomes the aforementioned problem of the prior art i.e.
the lack of compatibility of the desired individual components of a complex carrying nucleic acid synthesis activity.

' - 19-Without wanting to be limited thereto, this results in at least the following three approaches relating to the formation of such a chimeric protein carrying a nucleic acid synthesis activity.
The basic protein which forms the basis for the chimeric protein can be present in two basic forms. In the first form the basic protein only contains the nucleic acid synthesis activity but no sequence (also referred to herein as domain) which, in particular in vivo, can or could mediate an interaction between the nucleic acid synthesis activity and a processivity factor and especially not in such a manner that the interaction would lead to an increase in the processivity.
In a second form the basic protein contains a nucleic acid synthesis activity and additionally a sequence which, in particular in vivo, can or could promote an interaction between the nucleic acid synthesis activity and a processivity factor and especially in such a manner that the interaction increases the processivity.
The chimeric protein according to the invention can on the basis of the various forms of the basic protein be present in different fundamental embodiments.
(i) a first embodiment of the chimeric protein provides that the first form of the basic protein is used and that a sequence is attached thereto which mediates an interaction with a factor that increases the processivity of the nucleic acid synthesis activity. This attachment is typically carried out such that the interaction-mediating sequence adjoins the sequence of the nucleic acid synthesis activity and is optionally separated by a linker. Hence in this first embodiment of the chimeric protein the basic protein is supplemented by an interaction-mediating sequence.
(ii) In a second embodiment of the chimeric protein the second form of the basic protein is used. In this case the inherent sequence of the basic protein which mediates an interaction with a factor that increases the processivity of the nucleic acid synthesis activity is replaced by another sequence mediating an interaction with a factor increasing the processivity of the nucleic acid synthesis activity.
This other 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 in every case of a different origin. Consequently such a construction enables the nucleic acid synthesis activity of the basic protein to interact for the first time or to an increased extent with a factor that increases the processivity of the nucleic acid synthesis activity.
(iii) In a third embodiment of the chimeric protein the second form of the basic protein is used. In this case the inherent sequence of the basic protein which mediates an interaction with a factor that increases the processivity of the nucleic acid synthesis activity is supplemented by another sequence mediating an interaction with a factor increasing the processivity of the nucleic acid synthesis activity. This other 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 in every case of a different origin. Consequently such a construction enables the nucleic acid synthesis activity of the basic protein to interact for the first time or to an increased extent with a factor that increases the processivity of the nucleic acid synthesis activity.
For all embodiments of the chimeric protein mentioned above it can be said that the (amino acid) sequence of such a chimeric protein typically differs from the sequence of the basic protein. A further, but not obligatory, consequence can be that the complex that is formed from the chimeric protein having nucleic acid synthesis activity and the factor increasing the processivity is different from the complex of basic protein and factor increasing the processivity.
All proteins, polymerases and elongation proteins mentioned in the description are suitable as basic proteins, to the disclosure of which reference is herewith made.
The same applies to all other components in particular to those of the replication apparatus such as processivity factors, sliding clamp proteins and interaction-mediating or interaction-effecting sequences. Finally the definitions given in the introduction also apply to this part of the disclosure.
Although the nucleic acid synthesis activity can be derived from many organisms it is preferred that it is for example derived from the organism Carboxythermus hydrogenoformans (European Patent Application EP 0 834 569 Al) or one of the organisms such as e.g. Thermus aquaticus, Thermus caldophilus, Thermus chliarophilus, Thermus filiformis, Thermus Jlavus, Thermus oshimai, Thermus ruber, Thermus scotoductus, Thermus silvanus, Thermus species ZOS, Thermus species sp. 17, Thermus thermusphilus, Therotoga maritima, Therotoga neapolitana, Thermosipho africanus, Anaerocellum thermophilum, Bacillus caldotenax or Bacillus stearothermophilus.
If a sequence is mentioned herein, this usually refers to an amino acid sequence.
Nucleic acid sequences are usually referred to directly as nucleic acid sequences.
The various nucleic acids coding for the recombinant chimeric proteins according to the invention can be easily determined by persons skilled in the field by means of the genetic code and subsequently synthesized. Likewise suitable vectors for cloning and expressing the recombinant chimeric proteins according to the invention and methods for their preferably recombinant production are also known to persons skilled in the art (see for example Maniatis et al.; supra) Methods for producing antibodies, including monoclonal antibodies, which are directed against the recombinant chimeric proteins according to the invention are also known to persons skilled in the art.
The recombinant chimeric protein according to the invention can be used to elongate nucleic acids e.g. for the polymerase chain reaction, DNA sequencing, to label nucleic acids and for other reactions which include the in vitro synthesis of nucleic acids.
Hence a further subject matter of the present invention is a method for template-dependent elongation in which the nucleic acid to be elongated or at least a strand thereof is provided with at least one primer under hybridization conditions, the primer being sufficiently complementary to a part of or to a flanking region of the nucleic acid to be elongated and a primer elongation is carried out by a polymerase in the presence of nucleotides in which a recombinant chimeric protein according to the invention is used as the polymerase and additionally a sliding clamp protein is present in the reaction or in the reaction mixture in a preferred embodiment.

Methods for the template-dependent elongation of nucleic acids in which the elongation is initiated by a primer which has been hybridized to the template nucleic acid and has a free 3'-OH end available for the elongation are known to a person skilled in the art. In particular a polymerase chain reaction is carned out for the amplification. A double-stranded DNA sequence is usually used as the starting material of which it is intended to amplify a certain target region. Two primers are used for this which are complementary to the regions flanking the target sequence on each of the partial strands of the DNA double strand. However, in order to hybridize primers the DNA double strands are firstly denatured and in particular thermally melted. After the primer hybridization, an elongation is carried out by means of the polymerase, it is subsequently again denatured to separate the newly formed DNA strands from the template strands whereupon the nucleic acid strands formed in the first step are also available as a template together with the original template strands for a further elongation cycle, these are each again hybridized with primers and a new elongation takes place. This procedure is carried out in cycles with a thermal denaturation as an intermediate step.
The recombinant chimeric protein according to the invention can also be used for reverse transcription in which case either the protein according to the invention itself has reverse transcriptase activity or a suitable enzyme is additionally added which has reverse transcriptase activity irrespective of whether the thermostable in vitro complex has an inherent reverse transcriptase activity.
A recombinant chimeric protein according to the invention is also used for the reverse transcription of RNA into DNA which is preferred according to the invention in which case the nucleic acid synthesis activity of the protein has an inherent reverse transcriptase activity. This reverse transcriptase activity can either be the only polymerase activity or it can also be present together with an existing 5'-3' DNA polymerase activity. A preferred embodiment according to the invention of the recombinant chimeric protein contains the elongation protein derived from the organism Carboxydothermus hydrogenformans as disclosed in EP-A 0 834 569.
A further preferred use of the recombinant chimeric protein according to the invention is to sequence nucleic acids starting with at least one primer which is sufficiently complementary to a part of the nucleic acid to be sequenced in which again a template-dependent elongation is carried out or, in the case of RNA
sequencing, a reverse transcription using deoxynucleotides and dideoxynucleotides is carned out according to the method of Sanger. Within the framework of this preferred embodiment the respective derivatives described above are also suitable as deoxynucleotide and dideoxynucleotides. In particular it is preferable for the method according to the invention for elongating nucleic acids that the nucleic acids that are formed to be labelled. For this purpose it is possible to use labelled primers and/or labelled deoxynucleotides or/and labelled dideoxynucleotides and/or labelled ribo-nucleotides or appropriate derivatives thereof as already described above.
A further subject matter of the present invention is a method for labelling nucleic acids by inserting individual breaks in phosphodiester bonds of the nucleic acid chain and replacing a nucleotide at the breaks by a labelled nucleotide with the aid of a polymerase in which a thermostable in vitro complex according to the invention is used as the polymerase.
Such a method which is generally referred to as nick translation enables a simple labelling of nucleic acids. All aforementioned labelled ribonucleotides or deoxyribo-nucleotides or derivatives thereof are suitable for this provided they are accepted by the polymerase as a substrate.
The invention is further elucidated by the following examples and figures from which additional advantages and embodiments ensue.
Fig. 1 shows four sequence alignments of different elongation protein domains;
Fig.2 shows a schematic representation of the recombinant chimeric protein according to the invention;
Fig. 3 shows four consensus sequences of sliding clamp proteins;
Fig. 4 shows a table of the results of a yeast two-hybrid system;
Fig. 5, SA-C show alignments of several conserved regions of the sequence that is responsible for the interaction;

Fig. 6 A shows the entire sequence of one embodiment of a recombinant chimeric protein according to the invention;
Fig. 6 B shows a diagram of the basic construction of a chimeric protein according to the invention;
Fig. 7 shows the result of a polymerase chain reaction that was carried out using a chimeric protein according to the invention;
Fig. 8 A shows all fragments of Afn497 that can interact with the sliding clamp protein of Archaeoglobus fulgidus (Aft7335);
Fig. 8 B shows an alignment of C-terminal sequences of various genes from Archaeglobus fulgidus;
Fig. 9 shows the result of a yeast two-hybrid experiment which shows the interaction between an elongation protein and a sliding clamp protein and Fi. 10 shows the result of the amplification of genomic DNA using a recombinant chimeric protein according to the invention.
Example 1: Use of the yeast two-hybrid system to determine the amino acids that are important for the interaction between replication factors and sliding clamp protein The yeast two-hybrid system (Fields S., Song O., Nature 1989 Jul 20; 340 (6230):245-6) is used to determine the binding region of sliding clamp proteins (referred to herein in the following as SCP) and replication factors (referred to herein in the following as RF). The genes for SCPs and RFs are expressed in the vectors pGBT9 and pGAD424 (CLONTECH Laboratories, Inc.) to form fusion proteins having the DNA binding domains or the activation domains of GAL4:
DNA is purified by known methods from the organism Archaeoglobus fulgidus (DSM No. 4304). The microorganisms were cultured by the DSM ("Deutsche Sammlung fur Mikroorganismen"). Then the open reading frame of the genes are amplified by means of PCR from genomic DNA of Archaeoglobus fulgidus. The DNA obtained in this manner is cloned into the vectors pGBT9 and pGAD424.
Other vectors that are used in the two-hybrid system are also suitable for the method described in the following (these for example include pAD-GAL4-2.1, pBD-GAL4, pBD-GAL4 Cam, pCMV-AD, pCMV-BD, pMyr, pSos, pACT2, pAS2-1, pHISi, pLexA, pM, pHISi-1, pB42AD, pVPl6, pGADlO, pGBKT7, pLacZi, p8op-lacZ, pGAD GH, pGilda, pAD GL, pGADT7, pGBDU, pDBLeu, pPC86, pDBTrp, which are obtainable from CLONTECH Laboratories, Inc.). In addition modified clones are cloned into the same vectors. The modified clones are deletion mutations and mutations affecting individual amino acids or several amino acids of the SCP
and/or RF. Afterwards the ability of the modified clones to interact with one another in the two-hybrid system is measured. This enables domains or even individual amino acid residues to be determined that are important or essential for the interaction.
There are a number of methods for introducing deletions into a gene. One method for generating deletions is to prepare DNA fragments which contain the genes for SCP or RF. These fragments are obtained from a suitable vector either by PCR
or by restriction digestion. In addition genomic DNA is used from the organism from which the two proteins are derived. These various DNA fragments and the genomic DNA are reduced to small pieces by ultrasonic treatment and fragments which have lengths between the total length of the genes and about 100 bases are purified by preparative agarose gel electrophoresis. The ends of the fragments are filled in by treatment with a suitable enzyme (Klenow, Pwo-polymerase or others) or digested such that both strands have the same length and are thus blunt. These fragments are now incorporated by ligation into a pGAD424 and pGBT9 vector cleaved with SmaI
(or otherwise linearized and made blunt) or into other suitable vectors. This results in banks for numerous subfragments of the genes for SCP and RF which are each in both vectors of the two-hybrid system. The DNA of these banks is replicated and purified after transformation in Escherichia coli. In the next step the two banks are transformed into a suitable haploid yeast strain for the two-hybrid system such that the fusions of SCP or RF with the GAL4 DNA binding domains in one strain are present as a pairing type which is different from the fusion of RF or SCP and the GAL4 activation domain. Diploid cells in which the plasmids from the two banks are present in the same cell are now generated by pairing the two strains.
Suitable strains are PJ69-4 (James P, Halladay J, Craig EA, Genetics 1996 Dec; 144(4):
1425-36) or any other strain having a suitable genotype for the two-hybrid system.
Cells in which the reporter genes are activated are isolated and the plasmid DNA is isolated therefrom by preparation or the inserts are specifically amplified by PCR.
The sequences of the fusion fragments are determined by DNA sequence analysis.
The binding region (or the binding regions) is (are) determined by determining those regions in all clones (or which are always found in certain groups of clones) in which an interaction of the two fusion proteins comprising RF and SCP occurs.
This determination is carned out four times for each of the genes: once with the bank in the vector having the activation domain (preferentially: pGAD424) and once with the bank in the vector having the DNA binding domain (preferentially: pGTB9) and once with the total length clone of the binding partner and once with the gene bank of the fragments.
The described experiments enable minimum binding regions to be defined that can be utilized to construct recombinant chimeric proteins having an affinity for SCP.
Amino acid mutations are introduced for a more detailed characterization of the binding region and their effect on the binding characteristics is tested. This further narrows down the region and determines the positions in the protein involved in the binding. In a parallel preparation the positions involved in the binding are also determined by introducing random mutations in the binding region and analysing the effect on binding. In both cases it is determined whether the mutations destabilize the protein and thus have an indirect effect or whether they have no influence on the stability of the protein and thus have a direct effect.
The experimental procedures that are necessary for this are described in Maniatis et al., (Molecular Cloning(2"'~ edition, 3 volume set): A laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y. (1989)), in Ausubel et al (Current Protocols in Molecular Biology, John Wiley and Sons (1988)), in Abelson, J.N., and Simon, M.I. (editors) 1991 (Methods in Enzymology, volume 194, Guide to Yeast Genetics and Molecular Biology, Academic press) or in Adams, A. Gottschling, D.E.
Kaiser, CA, and Steams, T., 1997 (Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press). The handling of the two-hybrid system was carried out according to the instructions of the Clontech Company (yeast protocols handbook, PT3024-1).
Example 2: Further methods for mapping the binding region of SCP and RF
SCP and RF were obtained as recombinant proteins. In each case one of the two proteins was immobilized on a support and then the other protein was added in an unbound form in order to allow binding to the immobilized partner. Free i.e.
non-_ _27_ bound material was washed out and the bound protein was eluted for example by denaturation. The amount of bound protein is a measure for the binding (e.g.
used by Anderson D, Koch CA, Grey L, Ellis C. Moran MF, Pawson T, Science 1990 Nov. 16;250(4983): 979-82).
The analysis of deletion mutants of the proteins and mutants having a modified amino acid sequence allowed the mapping of the binding region. Deletions can be generated by proteolytic digestion or by the recombinant expression of deleted genes. In the same manner the binding region can be determined by co-immuno precipitation from cell extracts which contain the deleted forms of the proteins.
Competition of binding by peptides can give information on the sequence of the binding region.
A further approach for mapping the binding region is to prepare peptides and to measure the binding of the peptides to the other protein in each case. The sequence of the peptides can be random (Songyang Z, Prog Biophys Mol Biol. 1999; 71 (3-4):359-72) or be based on the amino acid sequence of the protein whose binding region is to be identified (peptide scans, Brix J, Rudiger S, Bukau B, Schneider-Mergener J, Pfanner N, J. Biol. Chem. 1999 Jun 4;274(23):16522-30). Such peptides can be prepared by chemical synthesis or by other methods such as phage display and be used for binding. An example is shown in fig. 3. In this case alignments of various consensus sequences were determined which are suitable for the preparation of such peptides.
A further method is to prepare antibodies against epitopes of the protein whose binding region is to be identified. If the antibody inhibits the binding, the epitope of the antibody overlaps with the binding region (e.g. Fumagalli S, Totty NF, Hsuan JJ, Courtneidge SA, Nature 1994 Apr 28;368(6474):871-4).
Finally another method for determining or mapping the binding region (interaction-mediating sequence) is to elucidate the fine structure of the complex using the methods of X-ray structural analysis, nuclear magnetic resonance or electron microscopy.

Example 3: Study of the interaction of proteins from Archaeoglobus fulgidus using the yeast two-hybrid system (Y2H) The coding regions of genes from Archaeoglobus fulgidus whose gene products can be used in vitro as an interaction-effecting sequence and/or nucleic acid synthesis activity were amplified by means of PCR, cloned into the vectors pGBT9 (vertical columns of fig. 9) and pGAD424 (horizontal rows of fig. 9) and expressed as hybrid proteins by gap repair in yeast PJ69-4a (for pGAD424) and PJ69-4alpha (for pGBT9). A positive control was also amplified by PCR, cloned into the vector pGBT9 and pGAD424 (see also horizontal rows of fig. 9) and expressed by gap repair in the yeast strain PJ69-4a (for pGAD424) and PJ69-4alpha (for pGBT9) as hybrid proteins. Diploid cells containing the two vectors were generated by pairing.
The expression of three independent reporters (HIS3, ADE2 and MEL 1 ) was measured. The expression of the HIS3 and ADE2 gene leads to a histidine or adenine prototrophy of the cells. Transcription of the MEL 1 gene leads to the production of the beta-galactosidase enzyme the activity of which can for example be detected by a colour reaction. In the experiment shown in fig. 9 the cells that grow in a histidine- and adenine-deficient medium are those which carry both vectors and in which additionally the expression products of these two vectors bind to one another. The interaction of the fusion proteins results in a reconstitution of a functional transcription factor that initiates the transcription of the reporter genes.
The production of the proteins coded on the reporter genes abolishes the histidine and adenine auxotrophy. This is due to the fact that the binding of the expression products initiates a transcription which makes it possible for the cells to grow.
All positive clones in this experiment were also positive with respect to the expression of the MEL 1 gene. The handling of the two-hybrid system is carried out according to the instructions of the Clontech Company (yeast protocol handbook, PT3024-1 ).
Example 4: Determination of the interaction-effecting sequence of Af0497 In order to determine the interaction-effecting sequence on the protein AfU497 (an elongation protein) which results in the interaction with the sliding clamp protein, ~- -29-the DNA of the gene for AfZ7497 was firstly obtained by restriction of a suitable vector which contained an elongation protein which contained an interaction-effecting sequence. This DNA was then fragmented by ultrasound and ligated into the two vectors pGAD424 and pGBDU. This resulted in banks of various fragments of the gene in the two vectors. Subsequently the yeast two-hybrid system was used to determine which of these fragments can result in an interaction with AfU335. For this purpose both banks were transformed in suitable yeast strains (pGAD424:
PJ69-4a, PGBDU: PJ69-4alpha). Diploid cells which contain the banks (AfU497) as well as the sliding clamp protein in a suitable vector for use in the two-hybrid system were obtained by pairing the yeast strains. All clones that activated the HIS2 reporter (see above) were isolated on plates without histidine and the inserts were selectively amplified by PCR of the yeast colony. The sequence of the inserts was determined and their position on the Afl7497 gene was determined. The result is shown diagrammatically in figure 8A: All found clones with the exception of one clone comprised the carboxyterminal end of the elongation protein which contains one or several interaction-effecting sequences. The smallest clone was composed of S 1 amino acids.
In order to determine whether the interaction-effecting sequence of the homologous protein of another organism is also present at the carboxyterminal end of the protein, the experiment was repeated using the polymerase protein and the sliding clamp protein from Pyrococcus horikoshi. For this purpose banks of the large subunit of the polymerase from P. horikosii were prepared and tested for interaction with the sliding clamp protein from P. horikosii by the method described above.
Again interacting fragments contained the carboxyterminal end of the elongation protein.
In order to check whether the interaction between the carboxyterminal end of the protein AfU497 and the sliding clamp protein is specific, it was tested whether the carboxyterminal protein fragment of AfiJ497 can also interact with other proteins from Archaeglobus fulgidus and other organisms. In no case was it possible to measure an interaction which indicates that the binding is very specific for the sliding clamp protein.

Thus the polymerase Taq does not bind to the SCP (PCNA from Archaeoglobus fulgidus) and no interaction whatsoever was measured in the yeast two-hybrid system. In order to test whether the carboxyterminal end of the protein AfU497 can result in an interaction with another protein to which it is fused, a fusion protein consisting of Taq and the S 1 carboxytenninal amino acids of AfU497 was prepared.
This protein was tested in the yeast two-hybrid system for interaction: As shown in fig. 4 the grafting of the interaction-effecting sequence of AfU497 to Taq results in a specific interaction of Tag with the sliding clamp protein Af1~335. The results of the corresponding Y2H experiments are shown in fig. 4.
This shows that the property of this fragment (the carboxyterminal end of the elongation protein Afl7497) to result in an interaction with the sliding clamp protein AfU335 can be grafted onto or transferred to another protein and in particular to another polymerase in order to result there in an interaction with the sliding clamp protein which is actually specific for Af0497.
An interaction with PCNA was measured with six proteins (see also fig. 5 and fig.
5B) from A. fulgidus. This shows that all these proteins contain similar interaction-effecting sequences that mediate the interaction with PCNA. Thus for example the polymerase delta large subunit (Af 0497, TREMBL number: 029753), the polymerase delta small subunit (Af 1790, TREMBL number: 028484), DP2 (Af 1722, TREMBL number: 028552), RPA2 (replication factor A), RFC2 (replication factor C) and PCNA (Af 0335, TREMBL number: 029912).
Such interaction-effecting sequences are contained in the last SO amino acids of the protein Afl7497. An examination of the proteins that interact with PCNA showed that all contain a motif that is located just before the carboxyterminal end of the amino acid sequence. Fig. 8B shows a list of the related sequences in which the conserved region is marked with a black bar.
This motif is also conserved in other organisms and genes. Fig. 5 shows the result of the search for such interaction-effecting sequences as well as the consensus sequences generated therefrom.

Example 5: Increasing the efficiency of a PCR by using PCNA as the SCP and a chimeric elongation protein according to the invention This example shows the influence of PCNA on the efficiency of a PCR reaction.
In this PCR reaction a 463 by fragment was amplified from plasmid DNA. The Taq fusion protein was used as the polymerase as also shown in fig. 6A. The reaction conditions for the PCR were as follows: 0.4 mM of each dNTP (pH 8.3) and 20 pmol of each primer in one reaction. A first primer (SEQ ID NO.: 13) having the sequence 5'-AGGGCGTGGTGCGGAGGGCGGT-3' and a second primer (SEQ 1D
NO.: 14) having the sequence 5'-TCGAGCGGCCGCCCGGGCAGGT-3' were used.
It turned out that the fusion of a 50 amino acid domain to the C-terminus of the native Taq DNA polymerase had no adverse effect on the polymerase properties since the Taq fusion protein per se already yields a product in a PCR
reaction. This domain can hence be used to mediate interaction between the sliding clamp protein PCNA from Archaeoglobus fulgidus and the Taq fusion protein and increase the efficiency of the PCR reaction due to its property as a processivity factor which is reflected in a considerably higher yield of PCR product. The result is also shown in fig. 7.
Example 6: Yeast two-hybrid system for detecting the interaction between the elongation protein Af 0497 of Archaeoglobus fulgidus and the sliding clamp protein Af 0335 of Archaeoglobus fulgidus Fig. 9 shows the results of a Y2H experiment in which row A contains cells which carry the empty pGAD424 vector (Clontech, Palo Alto, USA) such that a transcription activation domain is expressed, row B contains cells that carry the pGAD424 vector from which the Sacharomyces cerevesiae gene CDC48 is expressed as a fusion protein with the transcription activation domain; row C
contains cells which carry the pGAD424 vector from which the sliding clamp gene from Archaeoglobus fulgidus is expressed as a fusion protein with the transcription activation domain; row D contains no cells and raw E contains cells which carry the pGAD424 vector from which the elongation protein gene from Archaeoglobus fulgidus is expressed as a fusion protein with the transcription activation domain.
Column 1 contains cells which carry the empty pGBT9 vector (Clontech, Palo Alto, USA), column 2 contains cells which carry the pGBT9 vector from which the Saccharomyces cerevisiae gene UFD3 is expressed as a fusion protein with the DNA binding domain; column 3 contains cells which carry the pGBT9 vector from which the sliding clamp protein from Archaeoglobus fulgidus is expressed as a fusion protein with the DNA binding domain; column 4 contains cells which carry the pGBT9 vector from which the coupling protein from Archaeoglobus fulgidus is expressed as a fusion protein with the DNA binding domain and column 5 contains cells which carry the pGBT9 vector from which the elongation protein from Archaeoglobus fulgidus is expressed as a fusion protein with the DNA binding domain.
Example 7: Polymerase chain reaction of genomic DNA using a chimeric elongation protein Example 7 shows the influence of the sliding clamp protein on the efficiency of a PCR
reaction on long DNA fragments using the recombinant chimeric protein according to the invention. In this PCR reaction a 4954 by fragment was amplified from human genomic DNA. The recombinant chimeric protein according to the invention was used as the polymerase. The conditions correspond to the standard conditions of a PCR
reaction: pH 8.3, 0.4 mM of each pNTP and 20 pmol of each primer in one reaction. A
first primer (SEQ ID NO.: 15): S-AGGAACAACATATGACGCACTCT-3) and a second primer (SEQ ID NO.: 16): (5'-TAGGTGGCCTGCAGTAATGTTAG-3') were used.
It turned out that only the recombinant chimeric protein according to the invention having the sequence shown in fig. 6A allowed the generation of an unequivocal and specific PCR product under stimulation by the sliding clamp protein PCNA
whereas the same reaction mixture containing native Taq DNA polymerase yielded no defined product. This can be explained by the interaction of the sliding clamp protein with the recombinant chimeric protein according to the invention leading to . _ _33_ less dissociation of the recombinant chimeric protein according to the invention from the target DNA.
The figures which have been partially already referred to in the examples are described in more detail in the following.
All sequence alignments that are disclosed in the following figures were determined using the BLAST algorithm according to Altschul, S.F., Gish, W. Miller, W., Myers, E.W., and Lipman, D.J., J. Mol. Biol. 215, 403-410 (1990).
Fig. 1:
Fig. 1 shows sequence alignments of a total of four different elongation protein domains from various organisms i.e. for the elongation protein 1 from humans, Archaeglobus fulgidus, Methanococcus thermoautotrophicusm, PHBT (Pyrococcus horikoshii) and Methanococcus janashii. In the figures there is often a consensus sequence directly under the aligilment in which the variable positions are indicated by a single symbol. The amino acids in square brackets represent those amino acids expressed in the single letter code which can be present at the special position.
The amino acids are named according to the standard IUPAC single letter nomenclature and listed according to the prosite pattern description 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), also often written as "$" directly under the sequence S, T, N, Q, Y or C (amino acids with uncharged polar side chains), K, R, H, D or E (amino acids with charged and polar side chains) also often written as "&" directly under the sequence in addition X in the sequences or the sequence protocols denotes an arbitrary amino acid or insertion or deletion.
Fig. I shows four different consensus sequences for different regions of elongation proteins whereby for example the elongation protein 3 can be frequently found in ' ' -34-eubacteria, the elongation protein 4 also includes the Taq polymerase and can often be found in Pol I type polymerases. Hence the elongation protein 3 exhibits an alignment with a conserved region of the elongation protein from eubacteria and the consensus sequences derived therefrom. The following genes are shown:
DP3A ECOLI: DNA Pol III, alpha subunit, Escherichia coli, BB0579: DNA Pol III, alpha subunit, Borrelia burgdorferi, DP3A_HELPY: DNA Pol III, alpha subunit, Helicobacter pylori AASO: Aquifex aeolicus, section 50 and DP3A SALTY: DNA
Pol III, alpha subunit, Salmonella typhimurium).
Fig. 2:
Fig. 2 shows a diagram of a possible form of the recombinant chimeric protein according to the invention in which the sliding clamp, which is the factor increasing the synthesis activity of the polymerase, binds to the elongation protein carrying the nucleic acid synthesis activity via the domain containing the interaction-effecting sequence.
Fig. 3:
Fig. 3 shows four sliding clamp consensus sequences.
The following genes are shown for the sliding clamp re ion l: human PCNA as well as orthologues thereto from Archaeoglobus fulgidus, from Methanococcus janashii, from Pyrococcus horikoschii and from Methanococcus thermoautothrophicus.
Re '~ shows an alignment of a second conserved region of the sliding clamp from eukaryotes and Archae and the consensus sequences derived therefrom. The alignment was established using a second region of the sliding clamps that were already used above for region 1.
Re ig on 3 shows an alignment of a conserved region of the sliding clamp from eubacteria and the consensus sequences derived therefrom. The following genes are shown: AAPOL3B, DP3B ECOLI, S.TYPHIM, DP3B PROMI, DP3B PSEPU
and DP3B STRCO (AAPOL3B: Aquifex Aeolicus section 93: DP3B ECOLI:
DNA Pol III, beta chain, Escherichia coli, S.TYPHIM: DNA Pol III, beta chain, Salmonella typhimurium, P3B PROMI: DNA Pol III, beta chain, Proteus mirabilis DP3B PSEPU: DNA Pol III, beta chain, Pseudomonas putida DP3B STRCO:
DNA Pol III, beta chain, Streptomyces coelicolor).
Re ion 4 shows an alignment of a second conserved region of the sliding clamp from eubacteria (see organisms in region 3) and the consensus sequences derived therefrom.
Fig. 4.
Fig. 4 shows the interaction of chimeric proteins in the yeast two-hybrid system.
The result shown in fig. 4 proves that the property that promotes an interaction with the sliding clamp protein Af1~335 can be transferred to another protein and in particular to another polymerase where it results in an interaction with the sliding clamp protein. It is shown that the SCP binds itself and that the SCP binds a Taq fusion protein which contains an interaction-effecting sequence from Archaeoglobus fulgidus.
Fig. 5:
Fig. 5 shows three alignments of conserved regions of an interaction-effecting sequence from various organisms and for various genes:
Sequence 1 (polymerase gene, Af~497 homologue): Archaeoglobus fulgidus, Pyrodictium occultum, Aeropyrum pernix, Pyrococcus glycovorans, Pyrococcus furiosus, Thermococcus gorgonarius, Pyrococcus abyssi, Pyrococcus horikoshii, Thermococcus litoralis, Thermococcus fumicolans and Methanococcus jannaschii.

. . -36-Sequence 2 (polymerase gene, Af1722 homologue), Archaeoglobus fulgidus, Methanococcus jannaschii, Pyrococcus furiosus, Methanobacterium thermoauto-trophicum, Pyrococcus horikoshii and Pyrococcus abyssi and Sequence 3 (Af, 1347 homologue), Archaeoglobus fulgidus, Pyrococcus abyssi, Pyrococcus horikoshii, Methanobacterium thermoautotrophicum and Aeropyrum pernix.
The interaction-effecting sequences can be introduced into a chimeric protein according to the invention in order to bind a SCP.
Fig. 5B:
Fig. 5B shows two alignments of conserved regions of an interaction-promoting sequence from various organisms and for various genes:
Sequence 4 (RNAse gene, Af 0621 homologue): Archaeoglobus fulgidus, Pyrococcus abyssi, Pyrococcus horikoshii and Arabidopsis thaliana and Sequence 5 (polymerase gene): Archaeoglobus fulgidus, Pyrococcus abyssi, Metha-nococcus thermoautotrophicus, Methanococcus janashii, Mus musculus and Homo sapiens.
The interaction-effecting sequences can be introduced into a chimeric protein according to the invention in order to bind a SCP.
Fig. SC:
Fig. SC shows a list of characters that are used to label one or several different amino acids in which the amino acid or the property of the group is listed under "class" and the symbol used is listed under "key" and the amino acids in the group are listed under "residue".

_37_ Fig. 6A:
Fig. 6A shows the total sequence of a preferred embodiment of the recombinant chimeric protein according to the invention. In this case the elongation protein and thus the nucleic acid synthesis activity was derived from Thermus aquaticus and the' interaction-effecting sequence was derived from Archaeoglobus fulgidus.
Fig. 6B:
Fig. 6B shows a graphical overview of the construction of a preferred embodiment of the recombinant chimeric protein according to the invention. In this case the elongation protein is from Thermus aquaticus and the interaction-effecting sequence is from Archaeoglobus fulgidus.
Fig. 7:
Fig. 7 shows the use of a recombinant chimeric protein according to the invention in PCR; its sequence is shown in fig. 6A. In all lanes of this 1 % agarose gel one band can be seen having a size of 463 by for the amplificate of a PCR reaction initiated with plasmid DNA (pCR 2.1 vector including a 463 by fragment; Invitrogen, 9704 CH Groningen; Netherlands) which was carried out under the following conditions:
(95°C 5'denaturation; 30 x {95°C 30" denaturation; SS°C
30" hybridization; 72°C
40"elongation}; 72°C 7 additional elongations). 20 p1 of a 50 p.1 reaction mixture was applied to the gel. Lane 7 shows a size standard.
In lanes 2 (0.3 pg PCNA) and 3 (2.4 pg PCNA) it can be seen that the intensity of the bands increase compared to lane 1 (PCR reaction without PCNA) after adding increasing amounts of the sliding clamp protein (PCNA) as a stimulating agent at a constant Mg2+ ion concentration (2.5 mM). An additional increase in the intensity of the bands and hence an increased yield in the PCR reaction can be achieved at a higher Mgz+ ion concentration (3.5 mM) cf lane 4 (without PCNA) and lanes 5 (0.3 ug PCNA) and 6 (2.4 pg PCNA) with increasing amounts of PCNA.

i . _38_ Fig. 8A:
Diagram of all fragments of AfD497 (see above) that interact with the sliding clamp protein from Archaeoglobus fulgidus (Afn335). The arrows labelled "B"
represent clones that were found as a fusion with the DNA binding domain, the arrows labelled "A" represent clones that were found as a fusion with the activation domain.
Fig. 8B:
Fig. 8B shows an alignment of the C-terminal sequences of the following genes Af 0497-grUE, of 1195 Rfc, Af 0264 Rad2-Fenl, Af 0621 RNAseH, Acheaoglobus fulgidus.
Fig. 9:
Fig. 9 shows the result of a yeast two-hybrid test and proves a binding between the yeast proteins ufd3 and cdc48 (2B; positive control), between the elongation protein and the sliding clamp protein (3E; and in the reverse orientation SC), between the sliding clamp protein and the sliding clamp protein (3C) and between the coupling protein and the sliding clamp protein (4C). The fusion proteins are expressed from the vectors pGBT9 (vector) in column 1, pGBT9::ufd3 (positive control) in 2, pGBT9::PCNA in 3, pGBT9::klUE in 4, pGBT9::grUE in 5, pGAD424 (vector) in row A, pGAD424::cdc48 in B (positive control), pGAD424::PCNA in C, empty in D and pGAD::grUE in E.
Interactions of proteins from Archaeoglobus fulgidus were demonstrated with the Y2H system. The coding regions of genes from Archaeoglobus fulgidus whose gene products can be used in vitro were amplified in PCR, cloned into the vectors pGBT9 (vertical columns of fig. 27) and pGAD424 (horizontal rows of fig. 9) and expressed as hybrid proteins by gap repair in yeast PJ69-4a (for pGAD424) and PJ69-4alpha (for pGBT9). A positive control was also amplified by PCR, cloned into the vectors pGBT9 (see also vertical columns of fig. 9) and pGAD424 (see also horizontal rows of fig. 9) and expressed as hybrid proteins by gap repair in the yeast strain PJ69-4a (for pGAD424) and PJ69-4alpha (for pGBT9). Diploid cells containing the two vectors were generated by pairing according to the grid shown in fig. 9. The expression of three independent reporters (HIS3, ADE2 and MELD was measured. Fig. 9 shows the growth on medium without histidine and adenine. The cells which grow in this experiment are those that carry both vectors and where additionally the expression products of these two vectors bind to one another.
The binding of the expression products initiates transcription which leads to the abolition of the histidine and adenine auxotrophy.
All positive clones in this experiment were also positive with respect to the expression of the MEL 1 gene. The handling of the two-hybrid system was carned out according to the instructions of the Clontech Company (yeast protocol handbook, TP3024-1).
Fig. 10:
In all lanes of this 1 % agarose gel with the exception of lane 5 which shows a size standard, 20 p1 of a SO ~I preparation of the amplificate of a PCR reaction has been applied to the gel where the PCR was carried out on human genomic DNA having a size of 4954 base pairs (Roche Diagnostics, D-68305 Mannheim) under the following conditions: 95°C 5'denaturation; 35 x {95°C 30"
denaturation; 47°C 30"
hybridization; 72°C 12'elongation}; 72°C 10 additional elongations. In lane 3 (2.4 ~g PCNA) it can be seen that after adding increasing amounts of the sliding clamp protein (PCNA from Archaeoglobus fulgidus) to the recombinant chimeric protein complexes according to the invention as a stimulating agent one band is obtained at a constant Mgz+ ion concentration (6 mM) compared to lane 1 (PCR
reaction without PCNA) and lane 2 (0.3 pg PCNA), whereas it was not possible to generate a defined product in the PCR reaction at the same MgZ+ ion concentration with Taq DNA polymerase (lane 4).
The features of the invention disclosed in the present description and in the claims can be important individually as well as in any combination for the realization of the invention in its various embodiments.

Claims (43)

1. Recombinant chimeric protein comprising a) a first domain having nucleic acid synthesis activity and b) an interaction-mediating sequence characterized in that the interaction-mediating sequence results in the formation of a complex of nucleic acid synthesis activity and sliding clamp protein, said complex being different from the complex that the nucleic acid synthesis activity and/or the sliding clamp protein form(s) with their natural interaction partner(s) and the processivity of the nucleic acid synthesis activity is increased by the interaction-mediating sequence.
2. 2. Chimeric protein comprising a part having nucleic acid synthesis activity, said part being derived from a part having nucleic acid synthesis activity and a basic protein comprising an interaction-mediating part, and at least one interaction-mediating part wherein the interaction-mediating part is different from the interaction-mediating part of the basic protein and the interaction-mediating part mediates an interaction with the factor that increases the processivity of the nucleic acid synthesis activity.
3. 3. Chimeric protein as claimed in claim 2, characterized in that the protein contains the interaction-mediating part of the basic protein.
4. 4. Chimeric protein comprising a part having a nucleic acid synthesis activity, said part being derived from a basic protein which comprises a part having nucleic acid synthesis activity but no interaction-mediating part, and an interaction-mediating part, whereby the interaction-mediating part mediates an interaction with a factor that increases the processivity of the nucleic acid synthesis activity.
5. 5. Chimeric protein as claimed in one of the claims 2 to 4, characterized in that the factor is a sliding clamp protein.
6. 6. Recombinant chimeric protein as claimed in one of the claims 1 to 5, characterized in that the nucleic acid synthesis activity contains a consensus peptide sequence, the sequence being selected from the group comprising the sequences SEQ ID
   NO:1, 2 and 3.
7. 7. Recombinant chimeric protein as claimed in one of claims 1 to 6, characterized in that the sliding clamp protein contains a consensus peptide sequence, the sequence being selected from the group comprising the sequences SEQ ID NO.: 4, 5, 6 and 7.
8. 8. Recombinant chimeric protein as claimed in one of the claims 1 to 7, characterized in that the interaction-mediating sequence contains a consensus peptide sequence which is selected from the group comprising the sequences SEQ ID NO:8, 9, 10, 11 and 12.
9. 9. Recombinant chimeric protein as claimed in claim 8, characterized in that the interaction-mediating sequence contains a consensus peptide sequence according to SEQ m NO:8.
10. 10. Recombinant chimeric protein as claimed in one of the claims 1 to 5, characterized in that the interaction-mediating sequence is at the C-terminal end of the sequence carrying the nucleic acid synthesis activity.
11. 11. Recombinant chimeric protein as claimed in one of the claims 1 to 10, characterized in that a linker is located between the interaction-mediating sequence and the sequence carrying the nucleic acid activity.
12. 12. Recombinant chimeric protein as claimed in one of the claims 1 to 11, characterized in that the chimeric protein is thermostable.
13. 13. Recombinant chimeric protein as claimed in one of the claims 1 to 12, characterized in that the protein has a DNA polymerase activity.
14. 14. Recombinant chimeric protein as claimed in claim 13, characterized in that the protein has a 3'-5' exonuclease activity.
15. 15. Recombinant chimeric protein as claimed in one of the claims 1 to 14, characterized in that the protein has an RNA polymerase activity.
16. 16. Recombinant chimeric protein as claimed in one of the claims 1 to 13, characterized in that the protein has a reverse transcriptase activity.
17. 17. Recombinant chimeric protein as claimed in claim 13, characterized in that the incorporation rate of dNTPs and ddNTPs by the nucleic acid synthesis activity differs by a factor of less than 5.
18. 18. Complex comprising a) a recombinant chimeric protein as claimed in one of the claims 1 to 17 and b) a sliding clamp protein.
19. 19. Complex as claimed in claim 18, wherein the sliding clamp protein comprises a consensus peptide sequence which is selected from the group comprising the sequences according to SEQ ID NO: 4, 5, 6 and 7.
20. 20. Complex as claimed in claim 18 or 19 additionally comprising a nucleic acid.
21. 21. Nucleic acid coding for a recombinant chimeric protein as claimed in one of the claims 1 to 17.
22. 22. Vector containing a nucleic acid as claimed in claim 21.
23. 23. Vector as claimed in claim 22, wherein this is an expression vector.
24. 24. Cell containing a vector as claimed in claim 22 or 23.
25. 25. Use of a recombinant chimeric protein as claimed in one of the claims 1 to 17 to elongate nucleic acids.
26. 26. Use of a recombinant chimeric protein as claimed in one of the claims 1 to 17 to amplify nucleic acids.
27. 27. Use of a recombinant chimeric protein as claimed in one of the claims 1 to 17 for the reverse transcription of RNA into DNA.
28. 28. Use of a recombinant chimeric protein as claimed in one of the claims 1 to 17 to sequence DNA.
29. 29. Reagent kit for the elongation and/or amplification and/or reverse transcription and/or sequencing and/or labelling of nucleic acids which contains in one or several separate containers:
    
    a) a recombinant chimeric protein as claimed in one of the claims 1 to 17 and/or b) a complex as claimed in one of the claims 18 to 20 and preferably c) optionally at least one primer, buffer, nucleotides, cofactors and/or pyrophosphatase.
30. 30. Kit as claimed in claim 29, characterized in that it contains deoxynucleotides or/and derivatives thereof in addition to the substances a) and/or b) to amplify nucleic acids.
31. 31. Kit as claimed in claim 29 or 30, characterized in that it contains a DNA polymerase having 3'-5' exonuclease activity.
32. 32. Kit as claimed in claim 29, characterized in that it contains substances according to a) and/or b) which have a reverse transcriptase activity and preferably deoxynucleotides and/or derivatives thereof for reverse transcription.
33. 33. Kit as claimed in claim 29, characterized in that it contains dideoxynucleotides or/and derivatives thereof for sequencing in addition to deoxynucleotides or ribonucleotides or/and derivatives thereof.
34. 34. Method for the template-dependent elongation of nucleic acids in which the nucleic acid to be elongated or at least one strand thereof is provided with at least one primer under hybridization conditions, said primers being sufficiently complementary to a part of or to a flanking region of the nucleic acid to be elongated and the primer is elongated by a polymerase in the presence of nucleotides, characterized in that a recombinant chimeric protein as claimed in one of the claims 1 to 17 is used as the polymerase and preferably a sliding clamp protein is present in the reaction.
35. 35. Method for the amplification of a nucleic acid in which 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 part of or to a flanking region of the nucleic acid to be amplified and the primers are elongated by a polymerase in the presence of nucleotides characterized in that a chimeric protein as claimed in one of the claims 1 to 17 is used as the polymerase and a sliding clamp protein is preferably added to the reaction.
36. 36. Method as claimed in claim 35, characterized in that a polymerase chain reaction is carried out.
37. 37. Method as claimed in claim 36, characterized in that the reaction mixture contains two DNA polymerases of which at least one has a 3'-5' exonuclease activity and the 3'-5' exonuclease activity is either added to the reaction mixture by the recombinant chimeric protein or by an additional polymerase.
38. 38. Method as claimed in claim 37, characterized in that two recombinant chimeric proteins as claimed in one of the claims 1 to 17 are present in the reaction, wherein one is a protein as claimed in claim 13 and the other is a protein as claimed in claim 14.
39. 39. Method as claimed in claim 34, characterized in that a template-dependent elongation or reverse transcription is carried out according to the method of Sanger using deoxynucleotides and dideoxynucleotides or derivatives thereof in order to sequence nucleic acids starting with a primer that is complementary to a region that is adjacent to the nucleic acid to be sequenced.
40. 40. Method as claimed in claim 34, characterized in that labels are introduced during the elongation of the nucleic acids.
41. 41. Method as claimed in claim 40, characterized in that an agent is used which is selected from the group comprising labelled primers, labelled deoxynucleotides and derivatives thereof, labelled dideoxynucleotides and derivatives thereof and labelled ribonucleotides and derivatives thereof.
42. 42. Method for labelling nucleic acids by generating single breaks in phosphodiester bonds of the nucleic acid chain and replacing a nucleotide at the breakage points by a labelled nucleotide with the aid of a polymerase characterized in that a recombinant chimeric protein as claimed in one of the claims 1 to 17 is used as the polymerase.
43. 43. Antibody directed against a chimeric protein as claimed in one of the claims 1 to 17.