JPH11151087A - Dna polymerase gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the subject novel gene that comprises the DNA encoding the polypeptide of DNA polymerase having a specific amino acid sequence, is useful in the genetic engineering, and can produce DNA polymerase having excellent properties according to the purposes. SOLUTION: This novel gene comprises the DNA that encodes the polypeptide having the amino acid sequence represented by formula I, the DNA that encodes the polypeptide having the amino acid sequence resulting from deletion, substitution, insertion or addition of one or more amino acids from, with, into or to the amino acid sequence of formula I, the DNA having the base sequence represented by formula II, the DNA that has the base sequence resulting from deletion, substitution, insertion or addition of one or more bases from, with, into or to the base sequence of formula II, and the DNA that hybridizes with these DNAs and encodes the DNA polymerase having the activity of 3' →5' exonuclease. This gene is prepared by subjecting the genome DNA of Methanococcus jannaschii to the PCR using a partial sequence of the DNA polymerase gene followed by cloning.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel DNA polymerase gene and a novel DNA polymerase encoded by the gene. Further, the present invention relates to a method for producing the DNA polymerase.

[0002]

2. Description of the Related Art DNA polymerases known to date can be roughly classified into four families based on the common amino acid sequence. Among them, Escherichia coli D is generally used as a reagent for genetic engineering experiments.
NA polymerase I and thermophilic aquaticus D
It belongs to family A (pol-type enzyme) represented by NA polymerase and family B (α-type enzyme) represented by T4 phage DNA polymerase. When comparing the biochemical properties of each DNA polymerase in detail,
Although slightly different, DNA polymerases generally belonging to the same family have generally similar properties. Therefore, DNA polymerases having the most suitable properties among existing ones have been selected and used in accordance with experimental procedures.

[0003] For example, in WO 97/24444,
DN having strong 3 ′ → 5 ′ exonuclease activity, which is a proofreading function of DNA strand extension, and strong primer extension activity
A technology relating to A polymerase has been disclosed. A DNA polymerase having such properties is preferable as a DNA polymerase used in the PCR method, but a DNA polymerase having similar properties is not known.
Therefore, it is required to find a DNA polymerase having such properties in order to cope with various experimental operations.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a gene encoding a novel DNA polymerase which is useful in the field of genetic engineering and has excellent properties according to the purpose. It is a further object of the present invention to provide the novel DNA polymerase. It is a further object of the present invention to provide an expression vector containing the above gene, and a transformant transformed by the expression vector. It is a further object of the present invention to provide a method for producing the DNA polymerase of the present invention, which comprises culturing such a transformant.

[0005]

That is, the gist of the present invention is as follows: [1] (A) DNA encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, and (B) SEQ ID NO: 1. In the amino acid sequence shown,
DN encoding a polypeptide consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted or added
A, (C) DN consisting of the base sequence shown in SEQ ID NO: 7
A, (D) a DNA consisting of a base sequence in which one or more bases have been deleted, substituted, inserted or added in the base sequence shown in SEQ ID NO: 7, and (E) a DNA consisting of (A) to (D) A DNA selected from DNA that hybridizes to any DNA under stringent conditions, wherein the DNA encodes a polypeptide constituting a DNA polymerase having 3 ′ → 5 ′ exonuclease activity; [2] ( F) DNA encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2; (G) in the amino acid sequence shown in SEQ ID NO: 2,
DN encoding a polypeptide consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted or added
A, (H) DN consisting of the base sequence shown in SEQ ID NO: 8
A, (I) DNA consisting of a base sequence in which one or more bases are deleted, substituted, inserted or added in the base sequence shown in SEQ ID NO: 8, and (J) (F) to (I) A DNA selected from DNA that hybridizes to any DNA under stringent conditions, wherein the DNA encodes a polypeptide constituting a DNA polymerase having a 3 ′ → 5 ′ exonuclease activity;

[3] The DNA according to [1] and / or
Or an expression vector containing the DNA of [2], [4] a transformant transformed with the expression vector of [3], [5] a transformant of [4], 1]
A method for producing a DNA polymerase, which comprises culturing the DNA according to the above and / or the polypeptide encoded by the DNA according to the above [2] under conditions capable of expressing the DNA. [6] The DNA according to the above [1] A polypeptide constituting a DNA polymerase having 3 ′ → 5 ′ exonuclease activity; [7] a polypeptide encoded by the DNA according to [2], wherein → a polypeptide constituting a DNA polymerase having 5 ′ exonuclease activity, [8] a DNA polymerase comprising a polypeptide encoded by the DNA according to [1],
A DNA polymerase having 3 ′ → 5 ′ exonuclease activity,

[9] A DNA polymerase comprising the polypeptide encoded by the DNA according to [2],
A DNA polymerase having 3 ′ → 5 ′ exonuclease activity, [10] a DNA polymerase composed of the polypeptide according to [6] and the polypeptide according to [7], wherein the 3 ′ → 5 ′ exonuclease DNA polymerase having activity.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.

[0008] 1. DNA of the present invention Examples of the DNA of the present invention include the following. For example: 1) (A) DNA encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, (B) in the amino acid sequence shown in SEQ ID NO: 1,
DN encoding a polypeptide consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted or added
A, (C) DN consisting of the base sequence shown in SEQ ID NO: 7
A, (D) a DNA consisting of a base sequence in which one or more bases have been deleted, substituted, inserted or added in the base sequence shown in SEQ ID NO: 7, and (E) a DNA consisting of (A) to (D) DNA selected from DNA that hybridizes to any DNA under stringent conditions, and DNA encoding a polypeptide constituting a DNA polymerase having 3 ′ → 5 ′ exonuclease activity, 2) (F A) a DNA encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, (G) the amino acid sequence shown in SEQ ID NO: 2;
DN encoding a polypeptide consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted or added
A, (H) DN consisting of the base sequence shown in SEQ ID NO: 8
A, (I) DNA consisting of a base sequence in which one or more bases are deleted, substituted, inserted or added in the base sequence shown in SEQ ID NO: 8, and (J) (F) to (I) DNAs that hybridize to any of the DNAs under stringent conditions, such as DNAs encoding a polypeptide constituting a DNA polymerase having a 3 ′ → 5 ′ exonuclease activity, and the like. .

A DNA having the nucleotide sequence shown in SEQ ID NO: 7 and a DNA having the nucleotide sequence shown in SEQ ID NO: 8
Both NAs are found in the genomic DNA of the methanogenic archaebacterium Methanococcus jannaschii. These DNAs are derived from the non-α from the hyperthermophilic archaeon Pyrococcus furiosus.
It is a sequence showing homology to the nucleotide sequence of DNA encoding the polypeptide constituting the type and non-pol I type DNA polymerase (FIGS. 1, 2 and 3).

The amino acid sequence shown in SEQ ID NO: 1 and the amino acid sequence shown in SEQ ID NO: 2 are the base sequence shown in SEQ ID NO: 7 and the amino acid sequence shown in SEQ ID NO: 8, respectively.
Is an amino acid sequence derived from the base sequence shown in FIG.

Furthermore, as long as the DNA encodes a polypeptide constituting a DNA polymerase having 3 ′ → 5 ′ exonuclease activity, (B), (D),
In the amino acid sequence shown in SEQ ID NO: 1 and the amino acid sequence shown in SEQ ID NO: 2, such as the DNAs defined in (G) and (I), one or more amino acids are deleted, substituted, inserted or In the DNA encoding the polypeptide comprising the added amino acid sequence, and in the nucleotide sequence shown in SEQ ID NO: 7 and the nucleotide sequence shown in SEQ ID NO: 8, one or more bases are deleted, substituted, inserted or DNA comprising the added base sequence is also included in the DNA of the present invention. In this specification, “one or more” refers to one, or several or more.

Techniques for deleting, substituting, inserting or adding one or more bases and amino acids in a base sequence and an amino acid sequence include molecular cloning:
Various genetic engineering techniques described in Laboratory Manual Second Edition (1989, published by Cold Spring Harbor Laboratory, edited by T. Maniatis et al.).

Further, as long as the DNA encodes a polypeptide constituting a DNA polymerase having 3 ′ → 5 ′ exonuclease activity, the DNAs defined in (E) and (J) are also included in the DNA of the present invention. You. As used herein, the term “DNA that hybridizes under stringent conditions” refers to 0.5% SD together with a probe.
S, 0.1% bovine serum albumin (BSA), 0.1%
Polyvinylpyrrolidone, 0.1% Ficoll 400,
6 × SSC containing 1% denatured salmon sperm DNA (1 ×
SSC indicates 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0. 12) at 50 ° C
DNA that has hybridized with the probe after incubation for イ ン キ ュ ベ ー ト 20 hours.

2. 2. Polypeptide and DNA polymerase of the present invention The polypeptide of the present invention is a polypeptide constituting a DNA polymerase having 3 ′ → 5 ′ exonuclease activity, and 1) consists of the amino acid sequence shown in SEQ ID NO: 1. A polypeptide or a functional equivalent of the polypeptide (hereinafter referred to as "a first DNA polymerase constituent polypeptide"), or 2) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or a functional form of the polypeptide Equivalents (hereinafter referred to as "second DNA polymerase constituent polypeptide").

A specific example of the first polypeptide constituting the DNA polymerase is a polypeptide encoded by a DNA selected from the above (A) to (E), wherein 3 ′ →
A polypeptide constituting a DNA polymerase having 5 'exonuclease activity is exemplified.

Further, a specific example of the second DNA polymerase-constituting polypeptide is a polypeptide encoded by a DNA selected from the above (F) to (J),
Polypeptides constituting a DNA polymerase having 3 ′ → 5 ′ exonuclease activity.

Since the DNA polymerase activity and the 3 ′ → 5 ′ exonuclease activity appear in the presence of the two polypeptides of the present invention, the DNA of the present invention
It is presumed that the polymerase is constituted by non-covalently forming a complex of these two types of polypeptides.

Therefore, examples of the DNA polymerase of the present invention include the following.

1) At least one of the two polypeptides is a DNA polymerase which is a first DNA polymerase constituent polypeptide,
DNA polymerase having 3 ′ → 5 ′ exonuclease activity.

2) At least one of the two polypeptides is a DNA polymerase which is a second DNA polymerase constituent polypeptide,
DNA polymerase having 3 ′ → 5 ′ exonuclease activity.

3) A DNA polymerase comprising a first DNA polymerase constituent polypeptide and a second DNA polymerase constituent polypeptide, wherein 3 ′ →
DNA polymerase having 5 'exonuclease activity.

In the present specification, one unit of the DNA polymerase is determined as follows. A reaction solution containing a sample whose activity is to be measured [20 mM Tris-hydrochloric acid (p
H7.7), 15 mM MgCl ₂ , 2 mM 2-mercaptoethanol, 0.2 mg / mL activated DNA, 4 mM
0 μM dATP, dCTP, dGTP, dTTP, 6
0 nM [ ³ H] dTTP (Amersham)] 50 μL
Is incubated at 75 ° C. for 15 minutes. 40 μL thereof was spotted on DE paper (manufactured by Whatman), washed 5 times with a 5% Na ₂ HPO ₄ aqueous solution, and the radioactivity remaining on the DE paper was measured with a liquid scintillation counter. 10nm per 30 minutes
The amount of the enzyme that incorporates ol [ ³ H] dTMP into the substrate DNA is defined as one unit of the enzyme.

The 3 ′ → 5 ′ exonuclease activity can be detected as follows. pUC118
Single-strand DN using helper phage from plasmid
A is prepared and a 40-chain long D having a sequence complementary thereto is prepared.
NA (d40mer) is chemically synthesized. This d40me
After labeling the 5 'end of r with ³² P, it is mixed with pUC118 single-stranded DNA and annealed to obtain a template primer. A solution containing the protein to be measured and this template primer are added to a 20 mM Tris-HCl (pH 8.8) buffer containing ₂ mM MgCl ₂ and 2 mM 2-mercaptoethanol, and the mixture is incubated at 65 ° C. After several minutes, a reaction stop solution (95% formamide) is added, and the mixture is subjected to polyacrylamide gel electrophoresis containing 8 M urea, and 3 ′ → 5 ′ exonuclease activity is detected from the distribution of the labeled compound in the gel.

The "functional equivalent" described in this specification
Means the following. In naturally occurring proteins, in addition to polymorphisms and mutations in the gene encoding them, amino acid deletions, insertions,
It is known that mutations such as additions and substitutions can occur, but nevertheless, some exhibit substantially the same physiological activity and biological activity as a protein having no mutation. Those having no significant difference in their functions and activities even though they have structural differences are called functional equivalents. Here, the number of mutated amino acids is not particularly limited as long as they exhibit substantially the same physiological activity and biological activity. For example, one or more mutations (deletion, insertion, addition, etc.) , Substitution, etc.).

The same applies to the case where the above-described mutation is artificially introduced into the amino acid sequence of a protein. In this case, it is possible to prepare a further wide variety of mutants. For example, the methionine residue present at the N-terminus of a protein expressed in Escherichia coli is often said to be removed by the action of methionine aminopeptidase, but the removal is not completely performed depending on the type of protein, Both with and without methionine residues are produced. However, the presence or absence of this methionine residue often does not affect the activity of the protein. Also,
It is known that a polypeptide in which a certain cysteine residue in the amino acid sequence of human interleukin 2 (IL-2) is substituted with serine retains interleukin 2 activity [Science, Vol. 224, 1431 P. (1984)].

Further, when producing proteins by genetic engineering, they are often expressed as fusion proteins. For example, in order to increase the expression level of the target protein, an N-terminal peptide chain derived from another protein is added to the N-terminal, or an appropriate peptide chain is added to the N-terminal or C-terminal of the target protein for expression. The use of a carrier having an affinity for the peptide chain has facilitated the purification of the target protein. Therefore, even if a DNA polymerase having an amino acid sequence partially different from that of the DNA polymerase of the present invention, the protein is a “functional equivalent” as long as it exhibits essentially the same activity as the DNA polymerase of the present invention. ”Falls within the scope of the present invention.

Next, a method for producing the DNA polymerase of the present invention will be described.

For example, the DNA polymerase of the present invention is produced by culturing a transformant containing the expression vector containing the DNA of the present invention under conditions capable of expressing the polypeptide encoded by the DNA of the present invention. can do.

The conditions for culturing the transformant are not particularly limited, and may be any conditions under which the DNA of the present invention can be expressed. For example, as a vector, a lambda phage vector λSCREEN-1 and a Page Maker ^™
System PagePack Extract
In the case of a transformant obtained using [both manufactured by Novagen], the transformant is cultured in an LB medium containing ampicillin as a selective pressure, and at an appropriate time, IPTG (isopropyl-thio-β-D-galactoside). The expression may be induced by Transformants obtained using other vectors and hosts may be cultured in a medium under appropriate selection pressure under conditions suitable for protein expression. By culturing in this way, the desired protein is expressed.

The expressed protein can be recovered and purified by a protein purification method known in the art.

The expression of the DNA polymerase of the present invention can be confirmed, for example, by measuring the DNA polymerase activity using a crude extract prepared from the above transformant as a sample and using the above activated DNA as a substrate. it can.

The above expression vector and the above transformant which can be used for producing the DNA polymerase of the present invention are also included in the present invention.

The expression vector of the present invention comprises the DNA of the present invention.
An expression vector comprising: Such an expression vector can stably maintain the DNA of the present invention in a host such as Escherichia coli. Vectors used for the construction of such expression vectors include, for example, pET21a, pET21a
UC118, pWH5, pTV118, pSCREEN
-1b, etc., into which the DNA of the present invention can be inserted,
The vector is not particularly limited as long as it can be expressed. DNA
Is not particularly limited, and a known method such as a method using T4 DNA ligase can be used.

The transformant of the present invention is obtained by transforming with the expression vector of the present invention. Such a transformant can be obtained by introducing the expression vector of the present invention into a host. As a host, Escherichia coli, bacteria such as Bacillus, yeast such as Saccharomyces cerevisiae,
In addition to fungi such as Aspergillus and Pichia pastoris,
Animal cells such as insect cells, COS-7, and Vero cells can be used. Methods for introducing an expression vector into a host include, for example, the calcium phosphate method, CaCl ₂
Publicly known methods such as a method, a DEAE dextran method, and an electroporation method. Such a transformant can be selected by using, for example, a selectable marker of the expression vector used as an indicator. For example, a DNA that is extracted from the transformant and a probe that can specifically detect the DNA of the present invention are used. Can be used to confirm that the desired DNA has been introduced.

As a specific example of the transformant of the present invention, a transformant into which an expression vector having a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 7 has been introduced is used as a transformant of Escherichia coli.
A transformant into which Escherichia coli B into which BL21 (DE3) / pMJDP1 and an expression vector having a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 8 were introduced was used.
L21 (DE3) / pMJDP2 is a recombinant Escherichia coli designated and indicated, respectively. Such recombinant E. coli was sent to the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, 1997, 1-3 1-3 Higashi, Tsukuba, Ibaraki, Japan.
Escherichia coli BL21 (D
E3) / pMJDP1 as FERM P-16512 and Escherichia coli BL21 (DE3) / pM
JDP2 has been deposited as FERM P-16513.

As described above, since the DNA encoding the polypeptide constituting the DNA polymerase of the present invention has been cloned, a transformant into which these DNAs have been introduced is prepared to mass-produce the DNA polymerase of the present invention. It is possible to In this case, 1) the first DNA
DNA encoding a polymerase-constituting polypeptide;
A method of culturing a transformant containing both of the DNAs encoding the second DNA polymerase-constituting polypeptide and collecting the DNA polymerase from the culture;
DNA encoding NA polymerase constituent polypeptide
And a transformant containing a DNA encoding the second DNA polymerase-constituting polypeptide are separately cultured, and DN contained in the culture is cultivated.
A method such as a method of collecting a DNA polymerase by mixing the polypeptides constituting the A polymerase is included.

Here, the “transformant containing both the DNA encoding the first DNA polymerase constituent polypeptide and the DNA encoding the second DNA polymerase constituent polypeptide” includes the respective DNAs. May be co-transformed with two expression vectors, or may be a transformant prepared by incorporating both DNAs into one expression vector to express each polypeptide. Good.

[0038]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 (1) Preparation of Methanococcus yanashi genomic DNA Genome of Methanococcus yanashi DSM2661 ^T was converted to DSM (Deutsche Zamrunckvon microorganismen).
The cells were anaerobically cultured under the conditions specified by und Zellcurchulen (GmbH). About 52 mg of the cells obtained from a 500 mL culture were added to 1 mM EDTA, 100 mM
10 mL of 10 mM Tris-HCl containing NaCl (p
H8.0) After suspending in a buffer, 10% SDS was added for 1.5 hours.
mL was added. 20 mg / mL proteinase K
Was added and incubated at 55 ° C. for 60 minutes. After the suspension was subjected to phenol extraction and chloroform extraction, the obtained supernatant was subjected to ethanol precipitation to recover long-chain DNA. Transfer DNA to 1 mL TE
Buffer (10 mM Tris-HCl, 1 mM EDT
A, pH 8.0) and 250 mg / mL RNa
Then, 5 μL of s was added, and the mixture was incubated at 37 ° C. for 1 hour. This was subjected to a phenol extraction treatment and a chloroform extraction treatment, and the obtained supernatant was subjected to an ethanol precipitation treatment to collect DNA again. 660 by the above operation
μg of DNA was obtained.

(2) Amplification of Target DNA Region by PCR The entire base sequence of the genomic DNA of Methanococcus yanashi has already been determined [Science No. 2].
73, pp. 1058-1078 (1996)], and each open reading frame is also given a name. However, for DNA polymerase, α
Only the open reading frame of type DNA polymerase is described.

When the genomic DNA of Methanococcus yanashi was examined in detail, it was found that Pyrococcus furiosus non-
An open reading frame showing sequence homology to DNA encoding α-type and non-pol I type DNA polymerase constituent proteins was found. However, on the genome of Pyrococcus furiosus, the open reading frames encoding these two constituent proteins are adjacent, whereas in Methanococcus yanashi, these two open reading frames are located far apart on the genome. Existed. These open reading frames are MJ0702 and MJ163
It was 0.

FIGS. 1, 2 and 3 show two types of proteins (PfuDP1, PfP) constituting non-α type and non-pol I type DNA polymerases derived from Pyrococcus furiosus.
FIG. 3 shows a diagram showing the results of comparison between the amino acid sequence of uDP2) and the amino acid sequence of a polypeptide encoded by an open reading frame derived from Methanococcus yanashi (MJ0702 and MJ1630), which shows homology to the protein.

From FIG. 1, FIG. 2 and FIG. 3, the amino acid sequence of PfuDP1 of about 69 kDa and the amino acid sequence of the polypeptide encoded by MJ0702 are about 40% common, and the amino acid sequence of PfuDP2 of about 143 kDa , The amino acid sequence of the polypeptide encoded by MJ1630 is about 60% common. However, its function is completely unknown.

Therefore, in order to clone the genes in these two open reading frames, four types of oligonucleotides represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 in the sequence listing were synthesized. did. Among these, a combination of the oligonucleotide shown in SEQ ID NO: 3 and the oligonucleotide shown in SEQ ID NO: 4, and a combination of the oligonucleotide shown in SEQ ID NO: 5 and the oligonucleotide shown in SEQ ID NO: 6 Was used as a set of primers. Using 20 pmol of the oligonucleotide, that is, 40 pmol in total for one set of primers, PCR was performed in a reaction system having a total volume of 50 μL using 1 mg of the genomic DNA prepared in the above (1) as a template.

Specifically, after incubating at 98 ° C. for 2 minutes, KOD DNA polymerase (TOYOB) was used.
O) was added 2.5 units and then 55 minutes at 94 ° C for 55 minutes.
Forty cycles of 0.5 minutes at 72 ° C and 0.5 minutes at 72 ° C were performed. Reaction solution 5 after completion of PCR
When the μL was subjected to agarose gel electrophoresis, a DNA band of the desired chain length (about 1800 base pairs, 3450 base pairs) was detected. About 1800 base pairs of DNA
Is the open reading frame MJ0702,
The DNA of about 3450 base pairs is the open reading frame MJ1630.

(3) Incorporation of the gene of interest into a vector The PCR reaction solution of the above (2) was subjected to an ultrafiltration filter Su.
After purification using prec-02 (TAKARA),
The restriction enzymes NdeI and BamHI were added to cut the DNA. An NdeI sequence at the 5 ′ end thus obtained,
A fragment having a BamHI sequence at the 3 'end was inserted into the vector pE.
An expression vector was obtained by insertion into the NdeI-BamHI site of T21a (TAKARA). The ligation reaction solution containing the expression vector was added to Escherichia coli JM109 competent cells (TAKARA), and the mixture was allowed to stand on ice to transform the host. Plasmids were recovered from the obtained transformants, and the target clones were selected by confirming their nucleotide sequences.

(4) Culture of Transformant and Preparation of Crude Extract The plasmid containing the open reading frame MJ0702 and the plasmid containing the open reading frame MJ1630 isolated in (3) above were named pMJDP1 and pMJDP2, respectively. And both were introduced into Escherichia coli BL21 (DE3) for expression. Escherichia coli BL21 (DE3) / p, a transformant into which pMJDP1 has been introduced
Escherichia coli BL21 (DE3) / pMJDP2, which was a transformant into which MJDP1 and pMJDP2 had been introduced, were inoculated into 5 mL of LB medium containing 100 μg / mL ampicillin and cultured at 37 ° C. When the turbidity of the culture was 0.6 (A ₆₀₀ ), the expression inducer isopropyl-β-D-thiogalactopyranoside (IPT
G) was added at a concentration of 1 mM, and the cells were cultured for an additional 5 hours.

After completion of the culture, cells are collected, and after collection, 150 μL
Buffer A (50 mM Tris-HCl, pH 8.0,
The obtained cells were suspended in 2 mM 2-mercaptoethanol, 10% glycerol) and disrupted by ultrasonication. The supernatant was recovered by centrifugation of the disrupted cells, and the resulting supernatant was subjected to a heat treatment at 80 ° C. for 15 minutes to inactivate the DNA polymerase possessed by the E. coli host. Centrifugation was performed again, and the supernatant was recovered to obtain a crude extract. As a control, Escherichia coli BL21 (DE3) transfected with the vector pET21a not containing the gene of interest.
, A similar extract was prepared.

(5) Measurement of DNA polymerase activity As a reaction solution, 2 mM MgCl ₂ , 2 mM 2-mercaptoethanol, 0.2 mg / mL calf thymus DNA
(Activated), 40 μM dNTP (3 nM [α ³² P] dCTP or 60 nM [methyl ³
H] dTTP) containing 20 mM Tris-HCl (p
H8.8) A buffer solution was prepared. 45 μL of this reaction solution
Was added with 5 μL of the crude extract obtained in (4) above,
Incubate at 5 ° C for 10 minutes. A part of the reaction solution after the incubation was spotted on a DE81 paper filter, and the filter was then applied to 5% Na ₂ HPO.
₄ Washed 5 times with aqueous solution. Then, the radioactivity remaining on the filter was measured by autoradiography and a liquid scintillation counter. 4 and 5 show the results.
Shown in

4 and 5 that the DNA polymerase activity appears when the polypeptides encoded by the open reading frames MJ0702 and MJ1630 are mixed.

(6) Measurement of 3 ′ → 5 ′ exonuclease activity A single-stranded DNA was prepared from the pUC118 plasmid using a helper phage, and a 40-chain DNA (d40mer) having a sequence complementary thereto was prepared. Chemically synthesized. d
After labeling the 5 'end of the 40mer with ³² P, pUC11
8 and mixed with single-stranded DNA, and annealed to obtain a template primer. 20 mM Tris-HCl (pH 8.8) containing ₂ mM MgCl ₂ and 2 mM _2- mercaptoethanol
The crude extract obtained in the above (4) and this template primer were added to the buffer, and incubated at 65 ° C. 2
After 5 minutes, 5 minutes, 10 minutes, and 15 minutes, samples were sampled in equal amounts, and after adding a reaction stop solution (95% formamide), the mixture was subjected to polyacrylamide gel electrophoresis containing 8 M urea. After the electrophoresis was completed, the gel was dried, and the reaction product was detected by autoradiography. Fig. 6 shows the results.
Shown in

FIG. 6 shows that the open reading frames MJ0702 and MJ163 of Methanococcus yanashi were obtained.
When both of the polypeptides encoded by 0 are mixed, 3 ′ → useful as a proofreading function together with DNA synthesis activity
It can be seen that 5 'exonuclease activity was also detected.

As described above, the DNA polymerase of the present invention has high homology to non-α-type and non-pol I type DNA polymerases derived from Pyrococcus furiosus, which have greatly different properties from existing DNA polymerases. It also has 3 ′ → 5 ′ exonuclease activity. DNA polymerase of the present invention and Pyrococcus
The non-α type, non-pol I type DNA polymerase from Juliosus is expected to form a new family that does not belong to any conventional family.

[0054]

The DNA polymerase gene of the present invention
It encodes a protein having 3 ′ → 5 ′ exonuclease activity, and is useful in the field of genetic engineering.
It is a gene encoding a DNA polymerase having excellent properties according to the purpose. Furthermore, the DNA polymerase of the present invention has high homology to non-α-type and non-pol I type DNA polymerases derived from Pyrococcus furiosus, which have greatly different properties from existing DNA polymerases, and is a new DNA polymerase family. Can be proposed.

[0055]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 594 Sequence type: amino acid Number of chains: single-chain Topology: linear Sequence type: peptide Sequence: Met Glu Ile Ile Asn Lys Phe Leu Asp Leu Glu Ala Leu Leu Ser 5 10 15 Pro Thr Val Tyr Glu Lys Leu Lys Asn Phe Asp Glu Glu Lys Leu 20 25 30 Lys Arg Leu Ile Gln Lys Ile Arg Glu Phe Lys Lys Tyr Asn Asn 35 40 45 Ala Phe Ile Leu Leu Asp Glu Lys Phe Leu Asp Ile Phe Leu Gln 50 55 60 Lys Asp Leu Asp Glu Ile Ile Asn Glu Tyr Lys Asp Phe Asp Phe 65 70 75 Ile Phe Tyr Tyr Thr Gly Glu Glu Glu Lys Glu Lys Pro Lys Glu 80 85 90 Val Lys Lys Glu Ile Lys Lys Glu Thr Glu Glu Lys Ile Glu Lys 95 100 105 Glu Lys Ile Glu Phe Val Lys Lys Glu Glu Lys Glu Gln Phe Ile 110 115 120 Lys Lys Ser Asp Glu Asp Val Glu Glu Lys Leu Lys Gln Leu Ile 125 130 135 Ser Lys Glu Glu Lys Lys Glu Asp Phe Asp Ala Glu Arg Ala Lys 140 145 150 Arg Tyr Glu His Ile Thr Lys Ile Lys Glu Ser Val Asn Ser Arg 155 160 165 Ile Lys Trp Ile Ala Lys Asp Ile Asp Ala Val Ile Glu Ile Tyr 1 70 175 180 Glu Asp Ser Asp Val Ser Gly Lys Ser Thr Cys Thr Gly Thr Ile 185 190 195 Glu Asp Phe Val Lys Tyr Phe Arg Asp Arg Phe Glu Arg Leu Lys 200 205 210 Val Phe Ile Glu Arg Lys Ala Gln Arg Lys Gly Tyr Pro Leu Lys 215 220 225 Asp Ile Lys Lys Met Lys Gly Gln Lys Asp Ile Phe Val Val Gly 230 235 240 Ile Val Ser Asp Val Asp Ser Thr Arg Asn Gly Asn Leu Ile Val 245 250 255 Arg Ile Glu Asp Thr Glu Asp Glu Ala Thr Leu Ile Leu Pro Lys 260 265 270 270 Glu Lys Ile Glu Ala Gly Lys Ile Pro Asp Asp Ile Leu Leu Asp 275 280 285 Glu Val Ile Gly Ala Ile Gly Thr Val Ser Lys Ser Gly Ser Ser 290 295 300 Ile Tyr Val Asp Glu Ile Ile Arg Pro Ala Leu Pro Pro Lys Glu 305 310 315 Pro Lys Arg Ile Asp Glu Glu Ile Tyr Met Ala Phe Leu Ser Asp 320 325 330 Ile His Val Gly Ser Lys Glu Phe Leu His Lys Glu Phe Glu Lys 335 340 345 Phe Ile Arg Phe Leu Asn Gly Asp Val Asp Asn Glu Leu Glu Glu 350 355 360 Lys Val Val Ser Arg Leu Lys Tyr Ile Cys Ile Ala Gly Asp Leu 365 370 375 Val Asp Gly Val Gly Val Tyr Pro Gly Gln Glu Glu Asp L eu Tyr 380 385 390 Glu Val Asp Ile Ile Glu Gln Tyr Arg Glu Ile Ala Met Tyr Leu 395 400 405 Asp Gln Ile Pro Glu His Ile Ser Ile Ile Ile Ser Pro Gly Asn 410 415 420 His Asp Ala Val Arg Pro Ala Glu Pro Gln Pro Lys Leu Pro Glu 425 430 435 Lys Ile Thr Lys Leu Phe Asn Arg Asp Asn Ile Tyr Phe Val Gly 440 445 450 Asn Pro Cys Thr Leu Asn Ile His Gly Phe Asp Thr Leu Leu Tyr 455 460 465 His Gly Arg Ser Phe Asp Asp Leu Val Gly Gln Ile Arg Ala Ala 470 475 480 Ser Tyr Glu Asn Pro Val Thr Ile Met Lys Glu Leu Ile Lys Arg 485 490 495 495 Arg Leu Leu Cys Pro Thr Tyr Gly Gly Arg Cys Pro Ile Ala Pro 500 505 510 Glu His Lys Asp Tyr Leu Val Ile Asp Arg Asp Ile Asp Ile Leu 515 520 525 His Thr Gly His Ile His Ile Asn Gly Tyr Gly Ile Tyr Arg Gly 530 535 540 Val Val Met Val Asn Ser Gly Thr Phe Gln Glu Gln Thr Asp Phe 545 550 555 Gln Lys Arg Met Gly Ile Ser Pro Thr Pro Ala Ile Val Pro Ile 560 565 570 Ile Asn Met Ala Lys Val Gly Glu Lys Gly His Tyr Leu Glu Trp 575 580 585 Asp Arg Gly Val Leu Glu Val Arg Tyr 590

SEQ ID NO: 2 Sequence length: 1139 Sequence type: amino acid Number of chains: single chain Topology: linear Sequence type: peptide Sequence: Met Ile Val Met Val His Val Ala Cys Ser Glu Asn Met Lys Lys 5 10 15 Tyr Phe Glu Asn Ile Val Asp Glu Val Lys Lys Ile Tyr Arg Ile 20 25 30 Ala Glu Glu Cys Arg Lys Lys Gly Phe Asp Pro Thrasp Glu Val 35 40 45 Glu Ile Pro Leu Ala Ala Asp Met Ala Asp Arg Val Glu Gly Leu 50 55 60 Val Gly Pro Lys Gly Val Ala Glu Arg Ile Arg Glu Leu Val Lys 65 70 75 Glu Leu Gly Lys Glu Pro Ala Ala Leu Glu Ile Ala Lys Glu Ile 80 85 90 Val Glu Gly Lys Phe Gly Asn Phe Asp Lys Glu Lys Lys Ala Glu 95 100 105 Gln Ala Val Arg Thr Ala Leu Ala Val Leu Thr Glu Gly Ile Val 110 115 120 Ala Ala Pro Leu Glu Gly Gle Ile Ala Asp Val Lys Ile Lys Lys Asn 125 130 135 Pro Asp Gly Thr Glu Tyr Leu Ala Ile Tyr Tyr Ala Gly Pro Ile 140 145 150 Arg Ser Ala Gly Gly Thr Ala Gln Ala Leu Ser Val Leu Val Gly 155 160 165 Asp Phe Val Arg Lys Ala Met Gly Leu Asp A rg Tyr Lys Pro Thr 170 175 180 Glu Asp Glu Ile Glu Arg Tyr Val Glu Glu Val Glu Leu Tyr Gln 185 190 195 Ser Glu Val Gly Ser Phe Gln Tyr Asn Pro Thr Ala Asp Glu Ile 200 205 210 Arg Thr Ala Ile Arg Asn Ile Pro Ile Glu Ile Thr Gly Glu Ala 215 220 225 Thr Asp Asp Val Glu Val Ser Gly His Arg Asp Leu Pro Arg Val 230 235 240 Glu Thr Asn Gln Leu Arg Gly Gly Ala Leu Leu Val Leu Val Glu 245 250 255 Gly Val Leu Leu Lys Ala Pro Lys Ile Leu Arg His Val Asp Lys 260 265 270 Leu Gly Ile Glu Gly Trp Asp Trp Leu Lys Asp Leu Met Ser Lys 275 280 285 Lys Glu Glu Lys Glu Glu Glu Lys Asp Glu Lys Val Asp Asp Glu 290 295 300 Glu Ile Asp Glu Glu Glu Glu Glu Ile Ser Gly Tyr Trp Arg Asp 305 310 315 Val Lys Ile Glu Ala Asn Lys Lys Phe Ile Ser Glu Val Ile Ala 320 325 330 Gly Arg Pro Val Phe Ala His Pro Ser Lys Val Gly Gly Phe Arg 335 340 345 Leu Arg Tyr Gly Arg Ser Arg Asn Thr Gly Phe Ala Thr Gln Gly 350 355 360 Phe His Pro Ala Leu Met Tyr Leu Val Asp Glu Phe Met Ala Val 365 370 375 Gly Thr Gln Leu Lys Thr Glu ArgPro Gly Lys Ala Thr Cys Val 380 385 390 Val Pro Val Asp Ser Ile Glu Pro Pro Ile Val Lys Leu Lys Asn 395 400 405 Gly Asp Val Ile Arg Val Asp Thr Ile Glu Lys Ala Met Asp Val 410 415 420 Arg Asn Arg Val Glu Glu Ile Leu Phe Leu Gly Asp Val Leu Val 425 430 435 Asn Tyr Gly Asp Phe Leu Glu Asn Asn His Pro Leu Leu Pro Ser 440 445 450 Cys Trp Cys Glu Glu Trp Tyr Glu Lys Ile Leu Ile Ala Asn Asn 455 460 465 Ile Glu Tyr Asp Lys Asp Phe Ile Lys Asn Pro Lys Pro Glu Glu 470 475 480 Ala Val Lys Phe Ala Leu Glu Thr Lys Thr Pro Leu His Pro Arg 485 490 495 Phe Thr Tyr His Trp His Asp Val Ser Lys Glu Asp Ile Ile Leu 500 505 510 Leu Arg Asn Trp Leu Leu Lys Gly Lys Glu Asp Ser Leu Glu Gly 515 520 525 Lys Lys Val Trp Ile Val Asp Leu Glu Ile Glu Glu Asp Lys Lys 530 535 540 Ala Lys Arg Ile Leu Glu Leu Ile Gly Cys Cys His Leu Val Arg 545 550 555 Asn Lys Lys Val Ile Ile Glu Glu Tyr Tyr Pro Leu Leu Tyr Ser 560 565 570 Leu Gly Phe Asp Val Glu Asn Lys Lys Asp Leu Val Glu Asn Ile 575 580 585 Glu Lys Ile Leu Glu SerAla Lys Asn Ser Met His Leu Ile Asn 590 595 600 Leu Leu Ala Pro Phe Glu Val Arg Arg Asn Thr Tyr Val Tyr Val 605 610 615 Gly Ala Arg Met Gly Arg Pro Glu Lys Ala Ala Pro Arg Lys Met 620 625 630 Lys Pro Pro Val Asn Gly Leu Phe Pro Ile Gly Asn Ala Gly Gly 635 640 645 Gln Val Arg Leu Ile Asn Lys Ala Val Glu Glu Asn Asn Thr Asp 650 655 660 Asp Val Asp Val Ser Tyr Thr Arg Cys Pro Asn Cys Gly Lys Ile 665 670 675 Ser Leu Tyr Arg Val Cys Pro Phe Cys Gly Thr Lys Val Glu Leu 680 685 690 Asp Asn Phe Gly Arg Ile Lys Ala Pro Leu Lys Asp Tyr Trp Tyr 695 700 705 Ala Ala Leu Lys Arg Leu Gly Ile Asn Lys Pro Gly Asp Val Lys 710 715 720 Cys Ile Lys Gly Met Thr Ser Lys Gln Lys Ile Val Glu Pro Leu 725 730 735 Glu Lys Ala Ile Leu Arg Ala Ile Asn Glu Val Tyr Val Phe Lys 740 745 750 Asp Gly Thr Thr Arg Phe Asp Cys Thr Asp Val Pro Val Thr His 755 760 765 Phe Lys Pro Asn Glu Ile Asn Val Thr Val Glu Lys Leu Arg Glu 770 775 780 Leu Gly Tyr Asp Lys Asp Ile Tyr Gly Asn Glu Leu Val Asp Gly 785 790 795 Glu Gln Val Val Glu Leu Lys Pro Gln Asp Val Ile Ile Pro Glu 800 805 810 Ser Cys Ala Glu Tyr Phe Val Lys Val Ala Asn Phe Ile Asp Asp 815 820 825 Leu Leu Glu Lys Phe Tyr Lys Val Glu Arg Phe Tyr Asn Val Lys 830 835 840 Lys Lys Glu Asp Leu Ile Gly His Leu Val Ile Gly Met Ala Pro 845 850 855 His Thr Ser Ala Gly Met Val Gly Arg Ile Ile Gly Tyr Thr Lys 860 865 870 Ala Asn Val Gly Tyr Ala His Pro Tyr Phe His Ala Ala Lys Arg 875 880 885 Arg Asn Cys Asp Gly Asp Glu Asp Ser Phe Phe Leu Leu Leu Asp 890 895 900 Ala Phe Leu Asn Phe Ser Lys Lys Phe Leu Pro Asp Lys Arg Gly 905 910 915 Gly Gln Met Asp Ala Pro Leu Val Leu Thr Thr Ile Leu Asp Pro 920 925 930 Lys Glu Val Asp Gly Glu Val His Asn Met Asp Thr Met Trp Ser 935 940 945 Tyr Pro Leu Glu Phe Tyr Glu Lys Thr Leu Glu Met Pro Ser Pro 950 955 960 Lys Glu Val Lys Glu Phe Met Glu Thr Val Glu Asp Arg Leu Gly 965 970 975 Lys Pro Glu Gln Tyr Glu Gly Ile Gly Tyr Thr His Glu Thr Ser 980 985 990 Arg Ile Asp Leu Gly Pro Lys Val Cys Ala Tyr Lys Thr Leu Gly 995 1000 1005 Ser M et Leu Glu Lys Thr Thr Ser Gln Leu Ser Val Ala Lys Lys 1010 1015 1020 Ile Arg Ala Thr Asp Glu Arg Asp Val Ala Glu Lys Val Ile Gln 1025 1030 1035 Ser His Phe Ile Pro Asp Leu Ile Gly Asn Leu Arg Ala Phe Ser 1040 1045 1050 Arg Gln Ala Val Arg Cys Lys Cys Gly Ala Lys Tyr Arg Arg Ile 1055 1060 1065 Pro Leu Lys Gly Lys Cys Pro Lys Cys Gly Ser Asn Leu Ile Leu 1070 1075 1080 Thr Val Ser Lys Gly Ala Val Glu Lys Tyr Met Asp Val Ala Glu 1085 1090 1095 Lys Met Ala Glu Glu Tyr Asn Val Asn Asp Tyr Ile Lys Gln Arg 1100 1105 1110 Leu Lys Ile Ile Lys Glu Gly Ile Asn Ser Ile Phe Glu Asn Glu 1115 1120 1125 Lys Ser Arg Gln Val Lys Leu Ser Asp Phe Phe Lys Ile Gly 1130 1135

SEQ ID NO: 3 Sequence length: 40 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Sequence: GAAGGAGGAT ACATATGGAG ATAATAAATA AATTCTTAGA 40

SEQ ID NO: 4 Sequence length: 40 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Sequence: CTCTAAATTG GATCCTATTA ATATCTAACC TCTAAAACTC 40

SEQ ID NO: 5 Sequence length: 40 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Sequence: GCCGATTTAA GCATATGATT GTTATGGTTC ATGTTGCATG 40

SEQ ID NO: 6 Sequence length: 40 Sequence type: number of nucleic acid strands: single strand Topology: linear Sequence type: other nucleic acid Sequence: GACTGCAATG GATCCTATTA TCCTATCTTA AAGAAGTCAC 40

Sequence number: 7 Sequence length: 1782 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Sequence: ATGGAGATAA TAAATAAATT CTTAGATTTA GAGGCTTTAT TATCACCAAC TGTTTATGAA 60 AAGTTAAAAA ATTTTGATGA AGAAAAATTAAAG TAGAGAATTT 120 AAAAAATATA ACAACGCTTT TATTTTGTTA GATGAGAAGT TTTTAGACAT CTTTTTACAA 180 AAAGATTTGG ATGAAATAAT AAATGAATAT AAGGATTTTG ACTTTATATT TTACTACACT 240 GGAGAAGAAG AAAAAGAAAA ACCTAAAGAA GTAAAAAAAG AGATTAAAAA AGAAACTGAA 300 GAGAAAATAG AAAAAGAAAA AATAGAATTT GTTAAGAAAG AAGAAAAAGA ACAATTTATA 360 AAAAAATCTG ATGAAGATGT TGAAGAGAAA TTAAAACAAC TAATTTCCAA AGAAGAGAAA 420 AAAGAAGATT TCGATGCTGA GAGGGCTAAA AGATATGAAC ACATAACAAA AATAAAAGAG 480 AGTGTAAATA GTAGAATAAA ATGGATAGCT AAAGACATCG ATGCCGTGAT TGAGATATAT 540 GAAGATTCAG ACGTGTCTGG AAAATCCACA TGCACTGGAA CTATTGAGGA CTTCGTTAAA 600 TACTTTAGAG ACAGATTTGA AAGATTAAAG GTTTTTATTG AGAGAAAAGC TCAAAGAAAG 660 GGATATCCTC TAAAAGATAT AAAGAAAAT G AAAGGACAGA AGGATATTTT TGTCGTAGGA 720 ATCGTTAGTG ATGTTGATAG TACAAGAAAT GGGAACTTGA TAGTTAGGAT TGAAGACACC 780 GAAGATGAAG CAACGTTAAT TCTGCCAAAA GAAAAAATCG AAGCTGGAAA AATACCTGAC 840 GATATTTTGT TAGATGAAGT TATTGGAGCT ATTGGGACTG TTAGCAAATC TGGAAGTTCA 900 ATATACGTTG ATGAAATTAT ACGTCCAGCA TTACCACCAA AAGAACCAAA GAGAATTGAT 960 GAAGAGATAT ATATGGCATT TTTATCTGAT ATTCACGTTG GAAGTAAGGA GTTTTTGCAT 1020 AAAGAGTTTG AAAAATTCAT CAGATTTTTA AATGGAGATG TTGATAATGA ATTAGAGGAA 1080 AAGGTCGTTA GCAGATTAAA ATACATCTGC ATAGCTGGGG ATTTAGTTGA TGGGGTTGGT 1140 GTCTATCCAG GGCAGGAAGA GGATTTGTAT GAGGTAGATA TTATTGAGCA GTATAGAGAA 1200 ATAGCAATGT ATTTAGATCA GATTCCAGAG CATATAAGCA TAATCATCTC CCCAGGAAAC 1260 CACGATGCTG TTAGACCAGC AGAACCTCAA CCAAAACTGC CAGAGAAAAT AACTAAGCTA 1320 TTTAATAGAG ATAACATCTA CTTCGTTGGA AACCCATGCA CTCTCAACAT CCATGGCTTT 1380 GATACTCTAT TATATCACGG CAGAAGCTTT GACGACTTAG TTGGACAAAT AAGGGCTGCA 1440 AGTTATGAAA ATCCAGTAAC TATTATGAAG GAGTTGATAA AAAGAAGGCT ATTATGTCCA 1500 ACTTATGGAG GAAGATGTCC TATAGCTCCA GAACATAAA G ATTACTTGGT TATAGATAGG 1560 GATATTGATA TTTTACACAC TGGGCATATA CACATCAACG GTTATGGAAT TTATAGAGGA 1620 GTTGTTATGG TTAATAGCGG GACATTCCAA GAGCAAACAG ATTTCCAGAA GAGGATGGGT 1680 ATTAGCCCAA CACCTGCAAT AGTTCGATAG ATGAGGAT CATAG TAGAAGG

SEQ ID NO: 8 Sequence length: 3417 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Sequence: GTGATTGTTA TGGTTCATGT TGCATGCTCC GAAAATATGA AAAAGTATTT TGAAAACATT 60 GTTGATGAAG TTAAAAAGAAT TTACAAGAA GATATAGGATAG AGGTTTTGAC 120 CCAACTGATG AAGTTGAGAT TCCTTTAGCT GCTGATATGG CTGATAGAGT TGAGGGATTG 180 GTTGGGCCGA AAGGAGTAGC AGAGAGAATT AGAGAATTGG TTAAAGAGTT AGGTAAAGAA 240 CCAGCTGCAT TGGAGATAGC TAAAGAAATT GTTGAAGGAA AATTTGGAAA CTTTGATAAG 300 GAAAAAAAGG CAGAACAGGC AGTTAGAACT GCATTAGCTG TATTAACTGA AGGAATTGTT 360 GCTGCTCCAT TAGAGGGAAT TGCAGATGTT AAAATCAAAA AAAACCCAGA CGGAACTGAA 420 TATTTAGCTA TCTATTATGC AGGACCTATA AGAAGTGCTG GGGGAACTGC TCAAGCTCTA 480 TCTGTCTTAG TTGGAGATTT CGTAAGAAAG GCAATGGGTT TAGATAGATA CAAACCAACA 540 GAGGATGAGA TTGAGAGATA TGTTGAGGAG GTTGAGCTTT ACCAATCAGA AGTTGGGAGT 600 TTTCAATACA ACCCAACAGC AGATGAGATT AGAACAGCTA TAAGAAACAT CCCTATAGAG 660 ATTACTGGAG AAGCTACAGA TGATGTGGA A GTTTCAGGGC ATAGGGATTT GCCAAGGGTA 720 GAGACAAACC AACTGAGGGG AGGGGCTTTA TTAGTTTTGG TTGAGGGAGT TTTATTAAAA 780 GCTCCTAAAA TATTGAGGCA CGTTGATAAA TTAGGAATAG AGGGATGGGA CTGGCTTAAA 840 GATTTGATGA GTAAAAAAGA AGAAAAAGAG GAGGAAAAGG ATGAAAAAGT AGATGATGAA 900 GAAATAGATG AAGAGGAAGA AGAAATTAGC GGATACTGGA GAGATGTTAA AATAGAGGCA 960 AACAAAAAGT TTATAAGCGA AGTTATTGCT GGAAGGCCTG TTTTTGCCCA TCCATCAAAG 1020 GTTGGTGGAT TTAGGTTGAG ATATGGAAGG AGTAGAAACA CTGGTTTTGC TACTCAAGGA 1080 TTTCATCCTG CCTTAATGTA TTTGGTAGAT GAGTTTATGG CTGTTGGAAC CCAGCTAAAA 1140 ACTGAAAGGC CGGGAAAAGC TACATGTGTT GTGCCGGTTG ATAGCATTGA ACCACCAATT 1200 GTCAAGCTAA AAAATGGAGA TGTTATTAGA GTTGATACAA TAGAGAAAGC TATGGATGTT 1260 AGAAATAGGG TTGAGGAAAT TTTATTCTTA GGAGATGTTT TGGTTAATTA TGGGGATTTC 1320 TTAGAGAATA ATCACCCATT ATTGCCAAGT TGTTGGTGTG AGGAGTGGTA TGAGAAGATA 1380 TTGATAGCTA ATAATATAGA GTATGATAAG GATTTTATAA AGAACCCAAA GCCAGAGGAA 1440 GCTGTTAAGT TTGCTTTAGA AACAAAAACT CCACTACATC CAAGATTCAC CTATCACTGG 1500 CATGATGTTA GTAAGGAAGA TATAATCCTA TTAAGAAAT T GGTTGTTGAA AGGAAAAGAA 1560 GATAGCCTTG AAGGAAAAAA AGTTTGGATT GTTGATTTAG AGATAGAGGA AGATAAAAAA 1620 GCTAAAAGAA TCTTAGAATT AATTGGCTGC TGCCACTTAG TTAGAAATAA AAAGGTTATA 1680 ATTGAGGAGT ATTACCCTCT ACTCTACTCA CTGGGCTTTG ATGTTGAAAA TAAAAAGGAT 1740 TTAGTTGAAA ATATAGAGAA AATCTTAGAG TCAGCCAAAA ATAGTATGCA TCTTATAAAC 1800 TTATTAGCTC CGTTTGAAGT TAGAAGAAAC ACTTATGTAT ATGTTGGAGC AAGGATGGGA 1860 AGGCCAGAGA AAGCAGCACC AAGAAAGATG AAACCTCCAG TTAATGGTTT ATTCCCAATA 1920 GGTAATGCTG GAGGGCAAGT GAGATTGATA AACAAGGCAG TTGAGGAAAA CAATACAGAT 1980 GATGTTGATG TTTCTTACAC AAGATGTCCA AATTGTGGAA AAATTTCATT ATATAGAGTT 2040 TGCCCATTCT GTGGAACTAA GGTAGAGTTA GATAACTTTG GAAGAATTAA AGCTCCATTA 2100 AAAGATTATT GGTATGCCGC TTTAAAGAGA TTGGGTATAA ACAAGCCAGG AGATGTTAAG 2160 TGTATTAAAG GGATGACATC CAAGCAGAAG ATTGTTGAAC CATTAGAAAA AGCTATATTG 2220 AGGGCGATAA ATGAGGTTTA TGTCTTTAAA GACGGAACTA CAAGGTTTGA TTGCACAGAT 2280 GTGCCAGTAA CCCACTTTAA ACCAAATGAG ATAAACGTTA CTGTTGAAAA ATTGAGAGAG 2340 CTTGGCTATG ATAAAGATAT TTATGGCAAT GAGTTAGTTG ATGG GGAGCA GGTCGTTGAG 2400 CTAAAACCAC AAGATGTTAT CATCCCAGAG AGTTGTGCAG AGTATTTTGT TAAGGTAGCT 2460 AATTTTATAG ATGATTTATT GGAGAAGTTT TATAAAGTTG AAAGGTTTTA CAACGTAAAG 2520 AAAAAAGAGG ATTTAATTGG GCATTTAGTC ATTGGAATGG CTCCCCACAC ATCTGCTGGA 2580 ATGGTTGGAA GAATAATTGG TTATACAAAA GCAAATGTTG GTTATGCTCA TCCTTATTTC 2640 CATGCTGCAA AGAGAAGAAA CTGTGACGGG GACGAAGATT CTTTCTTTTT GCTATTAGAC 2700 GCGTTTTTGA ACTTCTCCAA AAAATTCCTA CCAGATAAGA GAGGAGGACA GATGGATGCC 2760 CCATTAGTCT TAACAACCAT ATTAGACCCA AAGGAAGTTG ATGGAGAAGT TCATAATATG 2820 GATACAATGT GGAGCTATCC ATTAGAGTTT TATGAAAAAA CCTTAGAAAT GCCTTCACCG 2880 AAAGAAGTTA AGGAGTTTAT GGAGACAGTT GAAGATAGAT TAGGAAAGCC AGAGCAGTAT 2940 GAAGGTATTG GCTATACTCA CGAAACATCA AGAATTGACT TAGGGCCGAA GGTTTGTGCT 3000 TATAAAACAT TAGGTTCAAT GTTAGAAAAA ACCACTTCCC AATTATCAGT TGCTAAGAAA 3060 ATTAGGGCTA CAGATGAAAG AGATGTTGCT GAGAAGGTTA TTCAATCCCA CTTCATCCCA 3120 GATTTAATTG GGAATTTAAG GGCTTTCTCA AGGCAGGCAG TTAGATGTAA ATGTGGAGCT 3180 AAGTATAGAA GAATACCTTT GAAAGGGAAG TGTCCAAAAT GTGGCTCTAA TTTAATATTA 3240 ACTGTCTCAA AGGGAGCTGT TGAGAAGTAT ATGGATGTTG CAGAGAAGAT GGCTGAGGAA 3300 TATAATGTAA ATGATTATAT AAAACAAAGAGA TTAAAGATTA TTAAAGAGGG GATTAATTCA 3360 ATATTTGAAA ATGAAAAAAG CAGACAGGTT AAGTTGATAG

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows non--derived from Pyrococcus furiosus.
Of the two proteins constituting the α-type and non-pol I type DNA polymerases, those of about 69 kDa (PfuDP
Showing homology to the amino acid sequence of 1) and the protein;
This is a comparison with the amino acid sequence of the polypeptide encoded by the open reading frame (MJ0702) derived from Methanococcus yanashi.

FIG. 2 shows non--derived from Pyrococcus furiosus.
Of the two proteins constituting the α-type and non-pol I type DNA polymerases, those of about 143 kDa (PfuD
FIG. 4 compares the amino acid sequence of P2) with the amino acid sequence of a polypeptide encoded by an open reading frame (MJ1630) derived from Methanococcus yanashi and showing homology to the protein.

FIG. 3 shows non--derived from Pyrococcus furiosus.
Of the two proteins constituting the α-type and non-pol I type DNA polymerases, those of about 143 kDa (PfuD
FIG. 4 compares the amino acid sequence of P2) with the amino acid sequence of a polypeptide encoded by an open reading frame (MJ1630) derived from Methanococcus yanashi and showing homology to the protein.

FIG. 4 shows that DNA polymerase activity appears when both polypeptides encoded by the open reading frames MJ0702 and MJ1630 of Methanococcus yanashi are mixed. This is the result of using autoradiography to detect that ³² P is incorporated into a DNA synthesis chain using [α ³² P] dCTP as a substrate. Lane 1 is Taq polymerase (0.
25 units), lane 2 is p
Spot when using a mixture of 3.5 μg of a crude extract of Escherichia coli (hereinafter referred to as HE1) into which MJDP1 has been introduced and 3.5 μg of a crude extract of Escherichia coli (HE3) into which only the pET21d vector has been introduced, lane 3 Is pMJDP2
3.5 E. coli crude extract (referred to as HE2)
In the case where a mixture of μg and 3.5 μg of HE3 was used, the spot in Lane 4 was 3.5 μg of HE1 and HE2.
Lane 5 is a spot when HE3 (7.0 μg) is used.

FIG. 5 is a graph showing the results of measuring the incorporation of ³ H into a DNA chain using [methyl ³ H] dTTP as a substrate by using a liquid scintillation counter.
Lanes 1 and 5 are data when H ₂ O was used instead of the reaction solution, lane 2 was data when HE3 (14 μg) was used, and lane 3 was HE1 (7.0 μg) and HE3 (7.0 μg). ), HEA (7.0 μg) and HE3 (7.
0 μg), and lane 6 shows HE1 (1.75 μg) and HE2 (1.75 μg).
In the case of using a mixture of
The data when a mixture of HE1 (3.5 μg) and HE2 (3.5 μg) was used. Lane 8 shows HE1 (5.25 μg).
μg) and HE2 (5.25 μg). Lane 9 shows HE1 (7.0 μg) and H2 (5.25 μg).
Data when a mixture of E2 (7.0 μg) was used, lane 10 was data when HE1 (7.0 μg) was used, and lane 11 was HE1 (7.0 μg) and HE2.
(1.75 μg), and lane 12 was HE1 (7.0 μg) and HE2 (3.
5 μg), and lane 13 shows HE1 (7.0 μg) and HE2 (5.25 μg).
In the case of using a mixture of
The data when using a mixture of E1 (7.0 μg) and HE2 (7.0 μg) are shown.

FIG. 6 shows the results of detecting the 3 ′ → 5 ′ exonuclease activity of the expression products of the open reading frames MJ0702 and MJ1630 of Methanococcus yanashi. The samples in lanes 1 to 4 are HE1
(7.0 μg) and HE3 (7.0 μg), the samples in lanes 5 to 8 were a mixture of HE2 (7.0 μg) and HE3 (7.0 μg), and lanes 9 to 12
The samples of HE1 (7.0 μg) and HE2 (7.0 μg)
g). Lane 13 shows the case where H ₂ O was used instead of the expression product. Lanes 1, 5, and 9 show cases where the incubation time was 2 minutes, and lanes 2, 6, and 1
0 is the case where the incubation time is 5 minutes, lanes 3, 7, and 11 are the cases where the incubation time is 10 minutes, and lanes 4, 8, and 12 are the cases where the incubation time is 15 minutes.
Minutes.

──────────────────────────────────────────────────続 き Continued on front page (51) Int.Cl. ⁶ Identification symbol FI (C12N 1/21 C12R 1:19) (C12N 9/12 C12R 1:19)

Claims (10)

[Claims] (1) a DNA encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1, (B) a DNA encoding the amino acid sequence represented by SEQ ID NO: 1,
DN encoding a polypeptide consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted or added
A, (C) DN consisting of the base sequence shown in SEQ ID NO: 7
A, (D) a DNA consisting of a base sequence in which one or more bases have been deleted, substituted, inserted or added in the base sequence shown in SEQ ID NO: 7, and (E) a DNA consisting of (A) to (D) A DNA selected from DNA that hybridizes to any DNA under stringent conditions, wherein the DNA encodes a polypeptide constituting a DNA polymerase having 3 ′ → 5 ′ exonuclease activity. 2. (F) a DNA encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2; (G) an amino acid sequence represented by SEQ ID NO: 2;
DN encoding a polypeptide consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted or added
A, (H) DN consisting of the base sequence shown in SEQ ID NO: 8
A, (I) DNA consisting of a base sequence in which one or more bases are deleted, substituted, inserted or added in the base sequence shown in SEQ ID NO: 8, and (J) (F) to (I) A DNA selected from DNA that hybridizes to any DNA under stringent conditions, wherein the DNA encodes a polypeptide constituting a DNA polymerase having 3 ′ → 5 ′ exonuclease activity. 3. An expression vector containing the DNA according to claim 1 and / or the DNA according to claim 2. A transformant transformed with the expression vector according to claim 3. 5. The transformant according to claim 4, wherein the transformant is
A method for producing a DNA polymerase, comprising culturing the DNA of claim 2 and / or a polypeptide encoded by the DNA of claim 2 under conditions that allow expression. 6. A polypeptide encoded by the DNA according to claim 1, which constitutes a DNA polymerase having 3 ′ → 5 ′ exonuclease activity. 7. A polypeptide encoded by the DNA according to claim 2, which constitutes a DNA polymerase having 3 ′ → 5 ′ exonuclease activity. 8. A DNA polymerase comprising the polypeptide encoded by the DNA according to claim 1, wherein the DNA polymerase has 3 ′ → 5 ′ exonuclease activity.
Polymerase. 9. A DNA polymerase comprising the polypeptide encoded by the DNA according to claim 2, wherein the DNA has 3 ′ → 5 ′ exonuclease activity.
Polymerase. 10. A DNA polymerase comprising the polypeptide according to claim 6 and the polypeptide according to claim 7, wherein the DNA polymerase has 3 ′ → 5 ′ exonuclease activity.