JP6326893B2 - Mutant PCNA - Google Patents

Description

The present invention relates to a DNA replication factor, and more particularly to a Proliferating Cell Nuclear Antigen (proliferating nuclear antigen) (also referred to herein as PCNA) having an excellent function of assisting DNA replication.

DNA replication is initiated when the double-stranded structure at the origin of replication is broken by a DNA helicase. Single-stranded DNA-binding protein binds to the unfolded DNA to stabilize the single strand, and primase for synthesizing a primer on each strand works. Next, replication factor Replication Factor C (also referred to herein as RFC) recognizes and binds to the primer and induces PCNA onto the DNA strand. PCNA serves as a clamp to keep the DNA polymerase on the DNA strand, and the DNA polymerase complexed with PCNA synthesizes the nascent strand.

As PCNA, those derived from Pyrococcus furiosus (hereinafter also referred to as Pfu-PCNA) and those derived from Thermococcus kodakaraensis KOD-1 (hereinafter also referred to as KOD-PCNA) are known. It is known that addition of Pfu-PCNA or KOD-PCNA to a DNA synthesis system promotes DNA extension activity (primer extension activity) and amplification enhancing activity of polymerase.

According to Patent Document 1, the PCNA multimer, which is one of the factors involved in DNA replication reaction, has an interface between the N-terminal region of one monomer and the C-terminal region of the other monomer. To be joined and formed. PCNA often forms trimers in eukaryotic cells and archaea. In wild-type Pfu-PCNA, the 139th, 143rd and 147th amino acid residue groups, and the 82nd, 84th and 109th amino acid residue groups in the amino acid sequence set forth in SEQ ID NO: 2 Are thought to form a network that joins and interacts with each other.

In Patent Document 1, a mutant of Pfu-PCNA is also studied. In wild-type Pfu-PCNA, a site that forms an intermolecular interaction in an interface region that contributes to multimer formation is composed of amino acid residues that attract each other in charge. On the other hand, in the mutant, the amino acid residues are formed so that the amino acid residues forming the intermolecular interaction repel each other in a charge, and remain in the monomer form or form a multimer. It is said to have amplification enhancing activity superior to that of the mold.

WO2007 / 004654 (International publication, republication)

To obtain a PCNA mutant having further excellent amplification enhancing activity in DNA replication.

Among the amino acid residues involved in PCNA multimer formation, the present inventors have determined that at least one of the amino acid residues present in the N-terminal region and at least one of the amino acid residues present in the C-terminal region are medium. By producing a mutant PCNA modified with a functional amino acid residue, the inventors have obtained the knowledge that it exhibits superior amplification-enhancing activity in DNA replication than in the past, thereby completing the present invention. The representative present invention is as follows.

[Claim 1]
In the amino acid sequence set forth in SEQ ID NO: 1 or 2, at least one of the amino acid residues present at the position corresponding to the group represented by (a) below, and at the position corresponding to the group represented by (b) A PCNA monomer comprising an amino acid sequence in which at least one of the amino acid residues is replaced with a neutral amino acid residue.
(A) 82, 84, 109th amino acid residue group (b) 139, 143, 147th amino acid residue group [Claim 2]
Item 5. The PCNA monomer according to Item 1, which is represented by any one of (1) to (4) below.
(1) A polypeptide comprising an amino acid sequence in which the amino acid residue corresponding to the 82nd position and the amino acid residue corresponding to the 143rd position in the amino acid sequence set forth in SEQ ID NO: 1 or 2 are replaced with neutral amino acid residues .
(2) A polypeptide comprising an amino acid sequence in which the amino acid residue corresponding to position 109 and the amino acid residue corresponding to position 143 in the amino acid sequence set forth in SEQ ID NO: 1 or 2 are replaced with neutral amino acid residues .
(3) In the PCNA monomer represented by (1) or (2), one or several amino acid residues at a site other than the group represented by (a) and (b) according to Item 1 A polypeptide comprising an amino acid sequence in which is substituted, deleted, inserted and / or added, and having amplification enhancing activity.
(4) It consists of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1 or 80% or more with the amino acid sequence shown in SEQ ID NO: 2, and has amplification enhancing activity Polypeptide.
[Section 3]
Item 3. A DNA encoding an amino acid sequence provided in the PCNA monomer according to Item 1 or 2.
[Claim 4]
A vector incorporating the DNA of Item 3.
[Section 5]
Item 5. A transformant comprising the vector according to item 4.
[Claim 6]
The transformant according to Item 5 is cultured in a medium, PCNA monomer and / or a multimer composed of the monomer is accumulated in the transformant or the medium, and the accumulated PCNA monomer A method for producing PCNA, comprising a step of recovering a polymer or a multimer.
[Claim 7]
Item 3. A DNA replication reagent comprising the PCNA monomer according to Item 1 or 2 and / or a multimer composed of the monomer.
[Section 8]
Item 8. A DNA replication kit comprising the reagent according to Item 7.
[Claim 9]
Item 9. The DNA replication kit according to Item 8, comprising a reagent for PCR.
[Section 10]
Item 3. A DNA replication method, wherein a DNA synthesis reaction is carried out in the presence of the PCNA monomer according to Item 1 or 2 and / or a multimer composed of the monomer and a DNA polymerase.
[Section 11]
Item 11. The DNA replication method according to Item 10, wherein the DNA synthesis reaction is PCR.

According to the present invention, a highly versatile DNA replication promoting factor excellent in amplification enhancing activity in DNA replication is provided.

Comparison of amplification enhancement activity of PCNA Comparison of amplification from blood with PCNA addition Comparison of PCR amplification in the presence of dUTP by adding PCNA Comparison of DNA polymerase synthesis rates by adding PCNA The figure which shows the amino acid region regarding multimer formation of PCNA

Hereinafter, the present invention will be described in more detail while showing embodiments of the present invention.
In this specification, the base sequence, amino acid sequence, and individual components thereof may be simplified by alphabetical symbols, but all follow the practices in the fields of molecular biology and genetic engineering. Further, in this specification, in order to simply show the mutation of the amino acid sequence, for example, a notation such as “D143A” is used. “D143A” indicates that the 143rd aspartic acid was substituted with alanine, that is, the type of amino acid residue before substitution, its location, and the type of amino acid residue after substitution. Further, the sequence numbers correspond to the sequence numbers described in the sequence listing unless otherwise specified. In the case of multiple mutants, the above notation is connected by “/”. For example, “D143A / D147A” indicates that the 143rd aspartic acid was substituted with alanine and the 147th aspartic acid was substituted with alanine.
Further, in this specification, the term “mutant type” in the case of “mutant PCNA” means that it has an amino acid sequence different from the conventionally known PCNA, and it is due to artificial mutation or due to mutation in nature. There is no distinction.

In addition, “the 82th“ corresponding ”amino acid in the amino acid sequence described in SEQ ID NO: 1” means 82 of SEQ ID NO: 1 in PCNA having an amino acid sequence that is not completely identical to the amino acid sequence shown in SEQ ID NO: 1. It is an expression including the amino acid sequence corresponding to the th.

In the present specification, in a amino acid sequence that is not completely identical to the amino acid sequence shown in SEQ ID NO: 1, a position (order) on SEQ ID NO: 1 and a corresponding position are when the primary structure of the sequence is compared (alignment) , A position corresponding to the position of SEQ ID NO: 1.
Various methods are known as methods for comparing the primary structures of sequences. For example, it can be calculated using an analysis tool that is commercially available or available through a telecommunication line (Internet).
In this specification, by using default (initial setting) parameters in ClustalW (http://clustalw.ddbj.nig.ac.jp/index.php?lang=ja) of DNA Databank of Japan (DDBJ), Compare the primary structure of the sequences.

  -PCNA of the present invention

One embodiment of the present invention includes at least one amino acid residue present in a position corresponding to the group represented by (a) below in the amino acid sequence set forth in SEQ ID NO: 1 or 2, and (b) A PCNA monomer comprising an amino acid sequence in which at least one of amino acid residues present at a position corresponding to the group represented by is replaced with a neutral amino acid residue.
(A) 82, 84, 109th amino acid residue group (b) 139, 143, 147th amino acid residue group

(1.1) Amino acid sequence SEQ ID NO: 1 is the amino acid sequence of PCNA derived from Thermococcus Kodakaraensis KOD1 strain (hereinafter also simply referred to as KOD). SEQ ID NO: 2 is an amino acid sequence of PCNA derived from Pyrococcus furiosus (hereinafter also simply referred to as Pfu).

In each of the amino acid sequences described in SEQ ID NOs: 1 and 2, amino acid residues involved in PCNA multimer formation are present in the N-terminal region and C-terminal region of each monomer. The PCNA multimer is formed by joining the N-terminal region of one monomer and the C-terminal region of the other monomer as an interface. In eukaryotic cells and archaea, PCNA often forms trimers. In the amino acid sequences represented by SEQ ID NOs: 1 and 2, the N-terminal region corresponds to the position of the group indicated by (a) above, and the C-terminal region corresponds to the position of the group indicated by (b) above. .

What has been described above and below using SEQ ID NO: 1 or SEQ ID NO: 2 as an example also applies to PCNAs other than the PCNAs whose sequences are specifically presented herein. For example, as shown in FIG. 5, in PCNA other than the PCNA shown in SEQ ID NOs: 1 and 2, (a) the 82, 84, 109th amino acid residue group of SEQ ID NO: 1 and (b) 139, 143, The region corresponding to the region related to multimer formation consisting of the 147th amino acid residue group is shown.

PCNA other than PCNA specifically presented herein includes, but is not particularly limited in origin, PCNA isolated from bacteria of the genus Pyrococcus and Thermococcus.
As PCNA derived from the genus Pyrococcus, Pyrococcus furiosus, Pyrococcus sp. Including, but not limited to, PCNA isolated from GB-D, Pyrococcus Wosei, Pyrococcus abyssi, Pyrococcus horikoshii.
PCNA derived from the genus Thermococcus includes Thermococcus kodakaraensis, Thermococcus gorgonaris, Thermococcus literalis, Thermococcus sp. JDF-3, Thermococcus sp. 9 degrees North-7 (Thermococcus sp. 9 ° N-7), Thermococcus sp. Including, but not limited to, PCNA isolated from KS-1, Thermococcus celler, or Thermococcus sicili.

In the PCNA monomer of the present invention, the combination of the amino acid substitution in the N-terminal region and the amino acid substitution in the C-terminal region is not particularly limited, but is preferably a PCNA monomer represented by the following (1) or (2) Is the body.
(1) A polypeptide comprising an amino acid sequence in which the amino acid residue corresponding to the 82nd position and the amino acid residue corresponding to the 143rd position in the amino acid sequence set forth in SEQ ID NO: 1 or 2 are replaced with neutral amino acid residues .
(2) A polypeptide comprising an amino acid sequence in which the amino acid residue corresponding to position 109 and the amino acid residue corresponding to position 143 in the amino acid sequence set forth in SEQ ID NO: 1 or 2 are replaced with neutral amino acid residues .

(3) In the PCNA monomer represented by the above (1) or (2), the above polypeptide is further represented by the above (a) and (b) as long as the polypeptide retains the amplification enhancing activity. A polypeptide having an amplification enhancing activity, comprising an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added at a site other than the group.
One or several mutations include restriction enzyme treatment, treatment with exonuclease, DNA ligase, etc., site-directed mutagenesis or random mutagenesis (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New (York) and the like can be carried out by introducing mutation into DNA encoding the PCNA monomer of the present invention described later. The variant PCNA monomer can also be obtained by other methods such as ultraviolet irradiation. Variant PCNA monomers also include naturally occurring variants (for example, single nucleotide polymorphisms) such as those based on individual differences in microorganisms holding PCNA, differences in species or genera.

(4) The above polypeptide is a polypeptide consisting of an amino acid sequence having an identity of 80% or more compared to the amino acid sequence shown in SEQ ID NO: 1 or 2 as long as the amplification enhancing activity is retained. Preferably, the identity between the amino acid sequence of the PCNA monomer of the present invention and the amino acid sequence shown in SEQ ID NO: 1 or 2 is 85% or more, more preferably 88% or more, still more preferably 90% or more. More preferably, it is 93% or more, more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more. Such a polypeptide comprising an amino acid sequence having a certain identity or more can be prepared based on the known genetic engineering techniques as described above.

Various methods are known as methods for calculating the identity of amino acid sequences. For example, it can be calculated using an analysis tool that is commercially available or available through a telecommunication line (Internet).
In the present specification, the homology algorithm BLAST (Basic local alignment search tool) http: //www.National Institute of Biotechnology Information (NCBI). ncbi. nlm. nih. By using default (initial setting) parameters in gov / BLAST /, amino acid sequence identity is calculated.

As PCNA as described in said (3) or (4), Preferably, the thing which changed the methionine corresponding to the 73rd in sequence number 1 or 2 to leucine (M73L) in order to increase the expression level of PCNA is mentioned. However, it is not limited to this.

(1.2) Neutral amino acid residue In the mutant PCNA monomer of the present invention, at least one of the amino acid residues at the positions shown above is substituted with a neutral amino acid residue. The kind of neutral amino acid is not particularly limited. Examples of neutral amino acids include glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, proline, serine, threonine, cysteine, methionine, asparagine, and glutamine. Preferably, alanine has the least influence on the three-dimensional structure of the peripheral site of the substitution site.

Specific examples of the mutant PCNA monomer of the present invention include R82A / E143A, R109A / E143A, etc., based on SEQ ID NO: 1. The case based on SEQ ID NO: 2 is the same as the case based on SEQ ID NO: 1, except that position 143 is D143A.

(1.3) Amplification enhancing activity

Amplification enhancing activity can be assessed by PCR. Amplification by adding PCNA to be evaluated to a PCR reaction solution containing DNA as a template, buffer, magnesium, dNTPs, primers, and DNA polymerase belonging to Family B, and comparing the amount of amplification with that without PCNA The enhancement activity can be confirmed. Preferably, the PCNA amplification enhancing activity is easy to evaluate in a PCR reaction system with a small amount of amplification, and more preferably, it is desirable to evaluate the PCNA amplification enhancing activity in a reaction system containing dUTP or reaction conditions with a short extension time. .

(1.3.1) Method for evaluating amplification enhancing activity In order to make it easier to evaluate the amplification enhancing activity of PCNA, the amplification enhancing activity of PCNA is compared in a reaction system in which the extension time is shortened and the amount of amplification is reduced.
In the present specification, the amplification enhancing activity is evaluated by the following method.
(I) PCR
Using 10 × PCR Buffer # 1 (concentrated to 10 times the concentration used in the reaction) attached to KOD DNA Polymerase (manufactured by Toyobo).
1 × PCR Buffer, and 1 mM MgCl ₂ ,
0.2 mM dNTPs,
6 pmol of primer (SEQ ID NO: 5 and 6),
50 ng of human genomic DNA (Roche),
0.4U KOD-Plus-
Prepare the reaction solution to contain
In 20 μl of the reaction solution, 300 ng of PCNA to be evaluated was added,
After 94 ° C. and 2 minutes of pre-reaction, PCR is performed with a schedule of 98 ° C., 5 seconds → 68 ° C., 36 seconds repeated 30 cycles.
(Ii) After completion of the PCR product analysis reaction, 5 μl of the reaction solution is subjected to agarose electrophoresis, stained with ethidium bromide, and amplified by comparing the amplified DNA fragment with that without addition of PCNA under ultraviolet irradiation. Activity can be evaluated. Addition of PCNA with high amplification enhancing activity increases the amplification amount.

(1.4)
In PCR, DNA replication is repeated using primers and template DNA, and DNA is amplified geometrically. Therefore, it is desirable that PCNA functions as a clamp for DNA polymerase, and quickly removes from the template after DNA polymerase is stabilized on the template or after amplification of a predetermined region.
The action and mechanism by which the PCNA mutant of the present invention can promote the DNA replication reaction than the conventional PCNA mutant is not necessarily clear, but the ring structure formed by the multimer is released when the temperature rises. It is presumed that the DNA replication reaction is smoothly repeated, and as a result, the DNA replication reaction is further promoted.

(1.5)
The PCNA monomer of the present invention is preferably isolated or purified. In addition, the PCNA monomer of the present invention may be present in a state dissolved in a solution suitable for the above storage or in a lyophilized state (for example, in powder form). “Isolated” when used in connection with the PCNA monomer of the present invention substantially includes components other than the enzyme (eg, contaminating proteins derived from host cells, other components, culture broth, etc.). No) states. Specifically, for example, the isolated PCNA monomer of the present invention has a contaminating protein content of less than about 20% by weight, preferably less than about 10%, more preferably less than about 5%. Even more preferably, it is less than about 1%. On the other hand, the PCNA monomer of the present invention may be present in a solution (eg, buffer) suitable for storage or measurement of enzyme activity. Moreover, one part or all part may form multimers, such as a trimer.

(2) Production method of mutant PCNA of the present invention (2.1) DNA encoding PCNA of the present invention
One embodiment of the present invention is DNA encoding an amino acid sequence provided in the above-described mutant PCNA of the present invention. Specifically, it is one of the following (A) to (D).

(A) In the amino acid sequence set forth in SEQ ID NO: 1 or 2, at least one of the amino acid residues present at the position of the group indicated by (a) below, and at the position of the group indicated by (b) DNA having a base sequence encoding an amino acid sequence in which at least one of the amino acid residues is replaced with a neutral amino acid residue.
(A) 82, 84, 109th amino acid residue group (b) 139, 143, 147th amino acid residue group

In the above (A), it preferably encodes an amino acid sequence in which positions 82 and 143 are replaced with neutral amino acid residues, or an amino acid sequence in which positions 109 and 143 are replaced with neutral amino acid residues. DNA having a base sequence to be

(B) In the nucleotide sequence represented by SEQ ID NO: 3 or 4, at least one of the nucleotides present in the position of the group represented by the following (a), and the nucleotide present in the position of the group represented by (b) At least one of them is DNA replaced with nucleotides encoding neutral amino acid residues.
(A) 244 to 246, 250 to 252 and 325 to 327th nucleotide groups (b) 415 to 417, 427 to 429, 439 to 441th nucleotide groups

In the above (B), preferably, DNA in which only 244th to 246th and 427th to 429th positions are replaced with nucleotides corresponding to triplets encoding neutral amino acid residues, or 325th to 327th and Only the 427th to 429th DNAs are replaced with nucleotides corresponding to triplets encoding neutral amino acid residues.

(C) The identity with the base sequence shown in (B) is 80% or more, preferably 85% or more, more preferably 88% or more, still more preferably 90% or more, still more preferably 93% or more, more preferably 95% or more, particularly preferably 98% or more, most preferably 99% or more of a nucleotide sequence, and at least one nucleotide present at the position of the group represented by (a) below: (b DNA which encodes a polypeptide having the above-described amplification enhancing activity, wherein at least one nucleotide present at the position of the group represented by (1) is replaced with a nucleotide encoding a neutral amino acid residue
(A) nucleotide groups 244 to 246, 250 to 252 and 125 to 327 (b) nucleotide groups 415 to 417, 427 to 429, and 439 to 441

In the above (C), the substitution to the nucleotide corresponding to the triplet encoding the neutral amino acid residue is only the 244th to 246th and 427th to 429th positions, or the 325th to 327th and 427th positions. It is preferable that only ˜429th.

(D) hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to the base sequence of the polynucleotide of SEQ ID NO: 3 or 4;
At least one of the amino acid residues present at the position of the group represented by (a) below and at least one of the amino acid residues present at the position of the group represented by (b) are neutral amino acid residues. Having a base sequence encoding the replaced amino acid sequence;
And the polynucleotide (a) 82, 84, 109th amino acid residue group (b) 139, 143, 147th amino acid residue group encoding PCNA having the amplification enhancing activity described above

In the above (D), substitution to nucleotides corresponding to triplets encoding neutral amino acid residues is only the 244th to 246th and 427th to 429th positions, or the 325th to 327th and 427th positions. It is preferable that only ˜429th.

As used herein, “DNA encoding a protein” refers to DNA from which the protein is obtained when it is expressed, that is, DNA having a base sequence corresponding to the amino acid sequence of the protein. Therefore, DNA that differs depending on codon degeneracy is also included.

As long as the protein having the amino acid sequence encoded by the DNA of the present invention has amplification enhancing activity, the homology with the nucleotide sequence shown in SEQ ID NO: 3 or 4 is 80% or more, preferably 85% or more. The nucleotide sequence is preferably 88% or more, more preferably 90% or more, still more preferably 93% or more, still more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more.

Various methods are known for calculating the identity of base sequences. For example, it can be calculated using an analysis tool that is commercially available or available through a telecommunication line (Internet).
In the present specification, nucleotide sequence homology is obtained by performing a search using blastn as a program and setting various parameters to default values in the homology algorithm Advanced BLAST 2.1 of the National Center for Biotechnology Information (NCBI). The value (%) is calculated.

The DNA of the present invention is a DNA that hybridizes under stringent conditions to a base sequence complementary to the base sequence shown in SEQ ID NO: 3 or 4 as long as the protein encoded by the protein has amplification enhancing activity. May be. Here, “stringent conditions” generally refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Such stringent conditions are known to those skilled in the art, and can be set with reference to, for example, Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York).

In this specification, “stringent conditions” refers to the following conditions.
50% formamide, 5 × SSC (0.15M NaCl, 15 mM sodium citrate, pH 7.0) as a hybridization solution, 1 × Denhardt solution, 1% SDS, 10% dextran sulfate, 10 μg / mL denatured salmon sperm DNA, 50 mM phosphate buffer (pH 7.5)) is used.

(2.2) Vector One embodiment of the present invention is a vector in which a DNA encoding the amino acid sequence of the mutant PCNA of the present invention is incorporated.

The vector of the present invention is a vector in which a DNA encoding the mutant PCNA of the present invention described in (2.1) above is incorporated. Here, the “vector” is a nucleic acid molecule (carrier) capable of transporting a nucleic acid molecule inserted therein into a target such as a cell, and can replicate the DNA of the present invention in an appropriate host cell. The type and structure are not particularly limited as long as it can be expressed. That is, the vector of the present invention is an expression vector. As the type of vector, an appropriate vector is selected in consideration of the type of host cell.
Specific examples of the vector include a plasmid vector, a cosmid vector, a phage vector, a virus vector (an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, a herpes virus vector, etc.) and the like. It is also possible to use a vector suitable for using a filamentous fungus as a host or a vector suitable for self-cloning.

When Escherichia coli is used as a host, for example, M13 phage or a modified form thereof, λ phage or a modified form thereof, pBlueScript or a modified form thereof, pBR322 or a modified form thereof (pBR325, pAT153, pUC8, etc.) may be used. it can. When yeast is used as a host, pYepSec1, pMFa, pYES2, etc. can be used. When insect cells are used as hosts, for example, pAc and pVL can be used. When mammalian cells are used as hosts, for example, pCDM8 and pMT2PC can be used, but the present invention is not limited thereto. .

Expression vectors usually contain a promoter sequence necessary for expression of the inserted nucleic acid, an enhancer sequence that promotes expression, and the like. An expression vector containing a selectable marker can also be used. When such an expression vector is used, the presence or absence of the expression vector (and the degree thereof) can be confirmed using a selection marker. Insertion of the DNA of the present invention into a vector, insertion of a selectable marker gene (if necessary), insertion of a promoter (if necessary), etc. are performed by standard recombinant DNA techniques (for example, Molecular Cloning, Third Edition, 1.84). , Cold Spring Harbor Laboratory Press, New York, which is a well-known method using restriction enzymes and DNA ligase).

(2.3) Transformant One of the embodiments of the present invention is a transformant comprising a vector incorporating a DNA encoding the amino acid sequence of the above-mentioned mutant PCNA of the present invention.
The means for introducing DNA into the host is not particularly limited. For example, the DNA is introduced into the host in a state of being incorporated into the vector described in (2.2) above. The host cell is not particularly limited as long as it can express the DNA of the present invention and produce mutant PCNA. Specifically, prokaryotic cells such as Escherichia coli and Bacillus subtilis, eukaryotic cells such as yeast, mold, insect cells, plant culture cells, and mammalian cells can be used.

When the host is a prokaryotic cell, examples include the genus Escherichia, the genus Bacillus, the genus Brevibacillus, the genus Corynebacterium, and the like, respectively, Escherichia coli (Escherichia) C600, Escherichia coli HB101, Escherichia coli DH5α, Bacillus subtilis, Brevibacillus choshinensis, Corynebacterium glutamicum, etc. Examples of the vector include pBR322, pUC19, and pBluescript.

When the host is yeast, the genus Saccharomyces, the genus Schizosaccharomyces, the genus Candida, the Pichia genus, the Cryptococcus genus, etc. (Saccharomyces cerevisiae), Schizosaccharomyces pombe, Candida utilis, Pichia pastoris, Cryptococcus p. Examples of the vector include pAUR101, pAUR224, and pYE32.

When the host is a filamentous fungal cell, examples include Aspergillus genus, Trichoderma genus, Colletotrichum genus, etc., respectively, Aspergillus oryzae, Aspergillus oryzae ), Trichoderma reesei, Colletotrichum Himalis, and the like. That is, in the transformant, exogenous DNA is usually present in the host cell, but a transformant obtained by so-called self-cloning using a microorganism from which the DNA is derived as a host is also a preferred embodiment.

The transformant of the present invention is preferably prepared by transfection using the expression vector shown in (2.2) above. Transformation may be transient or stable. Transfection can be performed using calcium phosphate coprecipitation method, electroporation, lipofection, microinjection, Hanahan method, lithium acetate method, protoplast-polyethylene glycol method, and the like.

The PCNA of the present invention produced in the transformant can be purified and isolated by a conventional method for protein purification, if necessary.

(2.4) Method for Producing PCNA In one embodiment of the present invention, the transformant is cultured in a medium, and the PCNA monomer or the monomer is used in the transformant or in the medium. A method for producing mutant PCNA, comprising the steps of accumulating the constituted multimer and recovering the accumulated monomer or multimer of PCNA.

The modified PCNA gene is transferred to an expression vector as necessary. For example, Escherichia coli as a host is transformed with the expression vector, and then applied to an agar medium containing a drug such as ampicillin to form colonies. The colony is inoculated in a nutrient medium such as LB medium or 2 × YT medium and cultured at 37 ° C. for 12 to 20 hours, and then the cells are crushed and the crude enzyme solution is extracted. A vector derived from pBlueScript is preferable. Any known method may be used as a method for crushing bacterial cells. For example, ultrasonic treatment, a physical crushing method such as French press or glass bead crushing, or a lytic enzyme such as lysozyme can be used. This crude enzyme solution is heat-treated at 80 ° C. for 30 minutes, centrifuged to remove host-derived protein, and subjected to SDS-PAGE, thereby confirming the expression of the target protein.

Any method may be used as a method for obtaining purified PCNA from the strain selected by the above method, for example, the following method. After collecting the cells obtained by culturing in a nutrient medium, the crude enzyme solution is obtained by crushing and extraction by enzymatic or physical crushing methods. The obtained crude enzyme extract is heat-treated, for example, at 80 ° C. for 30 minutes, and then the PCNA fraction is recovered by ammonium sulfate precipitation. This crude enzyme solution can be desalted by a method such as gel filtration using Sephadex G-25 (manufactured by Amersham Pharmacia Biotech). After this operation, the product is purified by subjecting it to Q Sepharose column chromatography to obtain a purified enzyme preparation. The purified enzyme preparation is purified by SDS-PAGE to such an extent that it shows an almost single band.

When obtaining this PCNA as a recombinant protein, it is preferable to change the methionine corresponding to position 73 in SEQ ID NO: 1 or 2 to leucine (M73L) in order to increase the expression level of PCNA.
In Patent Document 1, when natural Pfu-PCNA is prepared as a recombinant protein using Escherichia coli as a host, in addition to the translated protein from the original start Met, the N-terminus of about 20 kDa starts from the 73rd residue. Protein is produced as a by-product, but M73L suppresses the production of this protein of about 20 kDa, and Pfu-PCNA (M73L) produced in this way has properties that do not differ from wild-type PCNA. Therefore, M73L is treated as a quasi-wild type. Moreover, in the Example of patent document 1, M73L is handled as a quasi-wild type also about KOD-PCNA. For these reasons, the M73L mutation of the amino acid sequence described in SEQ ID NO: 1 or 2 is regarded as corresponding to the wild type in this specification.

If this PCNA is obtained as a recombinant protein, various modifications are possible. For example, if a DNA encoding the present PCNA and other appropriate DNA are inserted into the same vector and a recombinant protein is produced using the vector, the peptide consists of a recombinant protein linked to any peptide or protein. This PCNA can be obtained. In addition, modification may be performed so that addition of sugar chain and / or lipid, or processing of N-terminal or C-terminal may occur. By the modification as described above, it is possible to simplify the extraction / purification of the recombinant protein or add a biological function.

The PCNA monomer forms a multimer by joining the N-terminal region of one monomer and the C-terminal region of the other monomer as an interface. Since the conjugation is reversible, at least a part of those expressed as PCNA monomers may form a multimer.

(3) Utilization of Mutant PCNA of the Present Invention, etc. (3.1) DNA Replication Method One embodiment of the present invention is to carry out a DNA synthesis reaction in the presence of the above-mentioned PCNA and DNA polymerase of the present invention. This is a DNA replication method to be performed. In the above method, PCNA may be either a monomer or a multimer composed of the monomer, and both forms may be mixed.

The DNA replication method is not particularly limited. PCR, primer extension, nick translation, first strand cDNA synthesis by reverse transcriptase, and the like are mentioned, and DNA amplification by PCR is a preferred form.

The conditions for the DNA replication method are not particularly limited. For example, typical conditions in the case of PCR are shown below.
In addition to PCNA and DNA polymerase of the present invention, a DNA solution to be amplified, a primer, a DNA synthesis substrate (deoxynucleotide triphosphate (dNTP)), and a buffer solution containing Mg ions are mixed, set in a PCR device, and the PCR device Is set to the cycle shown by the following (I) to (IV) to carry out the reaction.
(I) The reaction solution is heated to about 94 ° C., and the temperature is maintained for 30 seconds to 1 minute to separate the double-stranded DNA into single strands.
(II) Rapid cooling to about 60 ° C. (which differs slightly depending on the primer), and annealing the single-stranded DNA and the primer.
(III) Heat again to a temperature range that is optimal for DNA polymerase activity without primer separation. Depending on the experimental purpose, the temperature is set to about 60-72 ° C. This temperature is usually maintained for 1 to 2 minutes depending on the time required for DNA synthesis and the length of amplification.
(IV) This is one cycle, and thereafter, the procedure from (I) to (III) is repeated to amplify a specific DNA fragment.

The PCNA of the present invention has good compatibility with various DNA polymerases and is highly versatile. A DNA polymerase is generally excellent in either extensibility or fidelity, and generally has advantages and disadvantages. However, by combining with the PCNA of the present invention, extensibility can be improved without reducing fidelity, so that DNA replication activity can be enhanced while reinforcing the disadvantages of DNA polymerase.
The PCNA of the present invention is particularly effective in improving extensibility and excellent in fidelity, but is particularly useful in combination with an α-type polymerase of a type inferior in extensibility. Typical α-type polymerases include those belonging to family B. Of these, those derived from archaea such as Pyrococcus and Thermococcus are preferred.
For example, Pyrobest DNA Polymerase (Takara Bio), TaKaRa EX Taq (Takara Bio), Vent DNA Polymerase (NEW ENGLAND BioLabs), Deep VentR DNA Polymer Pase (New EngT) ), KOD DNA Polymerase (Toyobo Co., Ltd.), and Pwo DNA Polymerase (Roche Diagnostics Co., Ltd.).

The α-type polymerase may be a mutant having reduced base analog detection activity.
Specific examples of such mutants include amino acid sequences related to the binding of uracil formed by the 1st to 40th and 78th to 130th amino acid sequences of DNA polymerase (SEQ ID NO: 10) derived from Thermococcus kodakaraensis KOD1 strain. Examples include DNA polymerase mutants that have been modified at least in one (uracil-binding pocket) and have reduced binding ability to uracil and inosine compared to wild-type DNA polymerase.

The amino acid sequence for uracil binding is highly conserved in DNA polymerases from Pyrococcus and DNA polymerases from Thermococcus. In the DNA polymerase (SEQ ID NO: 10) derived from Thermococcus kodakaraensis, it is formed by amino acids 1 to 40 and amino acids 78 to 130 as described above. In Pyrococcus furiosus (SEQ ID NO: 11), it is formed by amino acids 1 to 40 and amino acids 78 to 130. In Thermococcus gorgonarius (SEQ ID NO: 12), it is formed by amino acids 1 to 40 and amino acids 78 to 130. In Thermococcus litoralis (SEQ ID NO: 13), it is formed by amino acids 1 to 40 and amino acids 78 to 130. In Pyrococcus sp. GB-D (SEQ ID NO: 14), it is formed by amino acids 1 to 40 and amino acids 78 to 130. In Thermococcus sp. JDF-3 (SEQ ID NO: 15), it is formed by amino acids 1 to 40 and amino acids 78 to 130. In Thermococcus sp 9 ° N-7 (SEQ ID NO: 16), it is formed by amino acids 1 to 40 and amino acids 78 to 130. In Thermococcus sp. KS-1 (SEQ ID NO: 17), it is formed by amino acids 1 to 40 and amino acids 78 to 130. In Thermococcus cellar (SEQ ID NO: 18), it is formed by amino acids 1-40 and amino acids 78-130. In Thermococcus cyclis (SEQ ID NO: 19), it is formed by amino acids 1 to 40 and amino acids 78 to 130.

(1.2.2)
More preferred as an archaebacterial DNA polymerase mutant having reduced base analog detection activity for use in the nucleic acid amplification method of the present invention is assumed to be directly related to interaction with uracil, SEQ ID NO: 10 or an archaeal DNA polymerase mutant in which at least one of the amino acid sequence of SEQ ID NO: 11, 7, 36, 37, 90 to 97 and 112 to 119 is modified,
(A1) Poly consisting of an amino acid sequence having at least one amino acid modification among amino acids corresponding to amino acids 7, 36, 37, 90 to 97 and 112 to 119 of the amino acid sequence represented by SEQ ID NO: 10 or 11 It is a peptide.

The archaeal DNA polymerase mutant may be one represented by the following amino acid sequence (a2).
(A2) In the DNA polymerase mutant represented by (a1), at least one amino acid is further modified at positions other than the 7, 36, 37, 90 to 97, and 112 to 119th positions. 80% or more (preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 98% or more). More preferably, it is 99% or more) and has a reduced base analog detection activity.

The archaeal DNA polymerase mutant may be one represented by the following amino acid sequence (a3).
(A3) In the DNA polymerase mutant represented by (a1), one or several amino acids are further deleted, substituted, or added at sites other than positions 7, 36, 37, 90 to 97, and 112 to 119. And a polypeptide having reduced base analog detection activity.

Here, “several” is not limited as long as “decreased base analog detection activity” is maintained, but is, for example, a number corresponding to less than about 20% of all amino acids, preferably less than about 15%. It is a corresponding number, more preferably a number corresponding to less than about 10%, even more preferably a number corresponding to less than about 5%, and most preferably a number corresponding to less than about 1%. More specifically, the number of amino acid residues to be mutated is, for example, 2 to 160, preferably 2 to 120, more preferably 2 to 80, still more preferably 2 to 40, and even more. Preferably it is 2-5.

The “amino acids corresponding to positions 7, 36, 37, 90 to 97, and 112 to 119 in the amino acid sequence shown in SEQ ID NO: 10” are amino acid sequences that are not completely identical to the amino acid sequence shown in SEQ ID NO: 10. In the DNA polymerase having the amino acid sequence, the amino acid sequence relating to the binding of uracil corresponding to positions 7, 36, 37, 90 to 97, and 112 to 119 of SEQ ID NO: 10.

In the present specification, in the amino acid sequence that is not completely identical to the amino acid sequence shown in SEQ ID NO: 10, a certain position (order) on SEQ ID NO: 10 and the corresponding position are when the primary structure of the sequence is compared (aligned) , A position corresponding to the position of SEQ ID NO: 10.

In Patent Document 1 or 2, any of the amino acids at positions 7, 36, 37, 90 to 97, and 112 to 119, which are assumed to be directly related to the interaction with uracil, was modified. Several archaeal DNA polymerase mutants are exemplified, but not all variants have good properties to meet the challenges of the present application, some of which have lost activity .

(1.2.3)
More preferably, the archaeal DNA polymerase mutant having reduced base analog detection activity used in the nucleic acid amplification method of the present invention is the seventh, 36, or 93rd amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11. A polypeptide consisting of an amino acid sequence having at least one amino acid modification among the corresponding amino acids.

In the polypeptide, when the amino acid is modified at one position, it is preferable that tyrosine (Y) as the seventh amino acid is substituted with a nonpolar amino acid, specifically, Y7A, Y7G, Y7V, Y7L. An amino acid substitution selected from the group consisting of Y7I, Y7P, Y7F, Y7M, Y7W, and Y7C is exemplified. As another example, proline (P), which is the 36th amino acid, is preferably substituted with a positively charged polar amino acid, and specific examples include P36H, P36K, or P36R amino acid substitution. As another example, valine (V), which is the 93rd amino acid, is preferably substituted with a polar amine acid having a positive charge. Specifically, amino acid substitution of V93Q, V93K, or V93R is exemplified. .

More preferably, it is any one selected from the group consisting of Y7A, P36H, P36K, P36R, V93Q, V93K, and V93R.

In the polypeptide, when there are two amino acid modifications, preferred mutants include Y7A / V93K, Y7A / P36H, Y7A / P36R, Y7A / V93R, Y7A / V93Q or P36H / V93K. Preferred examples include Y7A / P36H or Y7A / V93K, but are not limited thereto.

The modified DNA polymerase used in the nucleic acid amplification method of the present invention may contain at least one amino acid modification in any of the amino acid sequences of the 3′-5 ′ exonuclease active region.
3′-5 ′ exoase activity refers to the ability to remove incorporated nucleotides from the 3 ′ end of a DNA polymer, and the above 3′-5 ′ exonuclease region is a DNA polymerase belonging to family B and highly DNA polymerase (SEQ ID NO: 10) derived from Thermococcus kodakaraensis, DNA polymerase (SEQ ID NO: 11) derived from Pyrococcus furiosus, DNA derived from Thermococcus gorgonarius Polymerase (SEQ ID NO: 12), DNA polymerase (SEQ ID NO: 13) derived from Thermococcus litoralis, DNA polymerase (SEQ ID NO: 14) derived from Pyrococcus sp. GB-D, derived from Thermococcus sp. JDF-3 DNA polymerase (SEQ ID NO: 15 DNA polymerase derived from Thermococcus sp 9 ° N-7 (SEQ ID NO: 16), DNA polymerase derived from Thermococcus sp KS-1 (SEQ ID NO: 17), DNA polymerase derived from Thermococcus cellar (sequence) No. 18), or DNA polymerase derived from Thermococcus cyclis (SEQ ID NO: 19), amino acids 137 to 147, 206 to 222, and 308 to 318.
The present invention is also applicable to DNA polymerases other than the DNA polymerase specifically presenting the sequence. In addition, in the archaeal DNA polymerase belonging to Family B other than the DNA polymerases shown in SEQ ID NOs: 10 to 19, 3′- consisting of amino acids 137 to 147, 206 to 222, and 308 to 318 of SEQ ID NO: 10 The region corresponding to the 5 ′ exonuclease region is shown.

The “amino acids corresponding to the 137th to 147th, 206th to 222nd, and 308th to 318th amino acids shown in SEQ ID NO: 10” are DNA polymerases having an amino acid sequence that is not completely identical to the amino acid sequence shown to SEQ ID NO: 10. This is an expression including amino acid sequences corresponding to positions 137 to 147, 206 to 222, and 308 to 318 of SEQ ID NO: 10.

The modification to the 3'-5 'exonuclease region may consist of substitution, deletion, or addition. Modifications to amino acids corresponding to positions 137 to 147, 206 to 222, and 308 to 318 in SEQ ID NO: 10 are shown.

In the DNA replication method of the present invention, since the PCNA of the present invention does not require RFC, it is not necessary to add RFC to the reaction system. RFC preparations are generally not commercially available, and their preparation is laborious. Even if the RFC standard can be used, it is necessary to set conditions such as an appropriate quantitative ratio with various factors of the DNA replication system other than the RFC when added and used. However, in the present invention, these are considered. There is a merit that it is not necessary. In particular, in the case of use in PCR, the PCNA of the present invention exhibits a promoting activity superior to that of the combination of wild-type PCNA and RFC, without using RFC together.

In addition, regarding the specific conditions of PCR, many manuals have already been issued, and in the method of the present invention, reaction conditions and the like may be appropriately adjusted with reference to those documents. Various conditions such as the amount of DNA polymerase added, PCR reaction time, reaction solution temperature setting, reaction solution components, reaction solution pH, and amount of template polynucleotide to be added are adjusted as PCR conditions. Is done.

In the method of the present invention, typical conditions in the case of PCR are shown below, but are not limited thereto.
In the amplification target DNA,
(A) DNA polymerase (b) PCNA
(C) a pair of primers in which one primer is complementary to the DNA extension product of the other primer (d) a DNA synthesis substrate (deoxynucleotide triphosphate (dNTP)), and
(E) mixing a buffer solution containing magnesium ions, ammonium ions and / or potassium ions;
By raising or lowering the temperature of the reaction solution using a thermal cycler or the like, a specific DNA fragment is amplified by repeating (1) DNA denaturation, (2) primer annealing, and (3) primer extension thermal cycle.
In the PCR method, BSA and a nonionic surfactant may be further used as necessary.

In the PCR method, if necessary, an antibody having the activity of suppressing the polymerase activity and / or 3'-5 'exonuclease activity of the thermostable DNA polymerase may be used. Examples of the antibody include a monoclonal antibody and a polyclonal antibody. This reaction composition is particularly effective for increasing the sensitivity of PCR and reducing nonspecific amplification.

(3.2) Reagent for DNA Replication, Kit for DNA Replication The reagent kit of the present invention is a reagent kit for DNA replication containing the PCNA of the present invention and other reagents as necessary. The PCNA of the present invention can be suitably used as a component of a DNA replication reagent in a form containing a monomer and / or a multimer composed of the monomer. The reagent kit of the present invention is particularly suitable as a reagent kit for PCR because the PCNA of the present invention can be used particularly preferably in PCR.

PCNA provided in the reagent kit of the present invention may be in any form, purified protein, recombinant polynucleotide incorporating a protein-encoding polynucleotide, or transformation into which this recombinant polynucleotide has been introduced. The form of a body etc. is illustrated. Preferred forms of the recombinant polynucleotide and transformant are as described above. Further, the PCNA of the present invention may be combined with another PCNA. In addition, when the PCNA of the present invention is provided in the form of recombinant DNA or transformant, reagents and the like used for expressing the PCNA of the present invention may be provided. Moreover, you may mix | blend the other components and medium generally used as a biotechnology reagent with the reagent containing PCNA of this invention as needed.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

Example 1
Preparation of KOD-PCNA mutant A plasmid containing a modified thermostable PCNA gene derived from Thermococcus kodakaraensis KOD1 strain was prepared.
As a DNA template used for mutagenesis, PCNA (SEQ ID NO: 3) (pKODPCNA) derived from Thermococcus kodakaraensis KOD1 strain cloned in pBlueScript was used. The mutation was introduced by using KOD-Plus-Mutageness Kit (manufactured by Toyobo) according to the instruction manual. The mutant was confirmed by decoding the base sequence. Escherichia coli DH5α was transformed with the obtained plasmid and used for enzyme preparation.

(Example 2)
Preparation of Pfu-PCNA mutant A plasmid containing a modified thermostable PCNA gene derived from Pyrococcus furiosus was prepared.
As a DNA template used for mutagenesis, PCNA (SEQ ID NO: 4) (pPfuPCNA) derived from Pyrococcus furiosus strain cloned into pBlueScript was used. The mutation was introduced by using KOD-Plus-Mutageness Kit (manufactured by Toyobo) according to the instruction manual. The mutant was confirmed by decoding the base sequence. Escherichia coli DH5α was transformed with the obtained plasmid and used for enzyme preparation.
The plasmids prepared in Example 1 and Example 2 are shown in Table 1.

(Example 3)
Preparation of modified heat-resistant PCNA The cells obtained in Example 1 and Example 2 were cultured as follows. First, 80 mL of TB medium (Molecular cloning 2nd edition, p.A.2) containing sterilized 100 μg / mL ampicillin was dispensed into a 500 mL Sakaguchi flask. Escherichia cultivated for 16 hours at 37 ° C. in 3 mL of LB medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride; Gibco) containing 100 μg / mL ampicillin in advance in this medium. E. coli DH5α (plasmid transformant) (using a test tube) was inoculated and cultured at 37 ° C. for 16 hours with aeration. The cells are collected from the culture solution by centrifugation, suspended in 50 mL of disruption buffer (30 mM Tris-HCl buffer (pH 8.0), 30 mM NaCl, 0.1 mM EDTA), and then subjected to sonication. By crushing, a cell lysate was obtained. Next, the cell lysate was treated at 80 ° C. for 15 minutes, and then the insoluble fraction was removed by centrifugation. Furthermore, nucleic acid treatment using polyethyleneimine, ammonium sulfate salting out, and Q sepharose chromatography were performed. Finally, a storage buffer (50 mM Tris-HCl buffer (pH 8.0), 50 mM potassium chloride, 1 mM dithiothreitol, 0 .1% Tween 20, 0.1% nonidet P40, 50% glycerin) to obtain modified heat-resistant PCNA.

Example 4
Evaluation of PCNA In order to compare the amplification enhancement activity of PCNA, the difference in amplification amount by PCR using mutants of KOD-PCNA (M73L, M73L / E143R, M73L / R109A / E143A, M73L / E143A / R82A) According to the method for measuring the amplification enhancing activity described in (1.3.1) above, a comparison was made by amplifying 3.6 kb of Human β-globin using PCR system GeneAmp9700 (Applied Biosystem). As described above, the M73L mutation is considered to correspond to the wild type.

FIG. 1 shows human β-globin DNA using five types of KOD-derived PCNA mutants (M73L, M73L / E143R, M73L / R109A / E143A, M73L / E143A / R82A) and KOD-Plus- as PCR enhancing factors. This is a result of electrophoresis of the obtained product by performing a 3.6 kb PCR reaction. In FIG. 1, lane 1 was added with KNA-Plus- without PCNA, and lanes 2-6 were added with K73-plus-mutated PCNA of M73L, M73L / E143R, M73L / R109A / E143A, and M73L / E143A / R82A, respectively. Is.

The results are shown in FIG. Although the amount of amplification was small without the addition of PCNA, improvement of the amount of amplification was confirmed by the addition of KOD mutant PCNA of M73L / R109A / E143A and M73L / E143A / R82A. On the other hand, addition of M73L and M73L / E143R resulted in inhibition of amplification.
PCNA forms a multimer and promotes the reaction, but if the multimer formation is too strong, it cannot be loaded into DNA without the action of RFC and the reaction does not proceed. The modification of M73L / R109A / E143A and M73L / E143A / R82A is a modification to the site involved in multimer formation. Since multimer formation was moderately weakened, PCNA can be loaded into DNA alone and the amount of PCR amplification is improved. It is possible that

For Pfu PCNA, M73L / D143R, M73L / R109A / D143A, M73L / D143A / R82A mutants were added to the above reaction, and M73L / R109A / D143A and M73L / D143A / R82A were amplified from M73L / D143R. It was confirmed that the amount was improved.

(Example 5)
In order to confirm the effect of adding amplified PCNA mutants from blood by adding PCNA, direct PCR from blood was performed. For comparison, KOD-PCNA mutants (M73L, M73L / R82A / E143A) were used, and the difference in the amount of HBg 3.6 kb was compared.

PCR was performed using KOD-Plus-Ver. 2 (Toyobo) attached Buffer, dNTPs, MgSO ₄ , enzyme solution (KOD-Plus-), 1 × PCR Buffer, and 1.0 mM MgSO ₄ , 15 pmol primer (SEQ ID NOs: 5 and 6), 1U In a 50 μl reaction solution containing 1 μg of KOD-Plus-, blood was added to 8% of the reaction solution, and no PCNA and various concentrations of PCNA variants (0.5 μg, 1 μg, 2 μg, 4 μg) Were compared. The reaction was performed using PCR system GeneAmp 9700 (Applied Biosystem) on a schedule of 94 cycles at 94 ° C. for 2 minutes followed by 30 cycles of 98 ° C., 10 seconds → 68 ° C. and 4 minutes.
After completion of the reaction, 5 μl of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amplification amount of the amplified DNA fragment was confirmed under ultraviolet irradiation.

In FIG. 2, blood is used as a sample, a reaction solution is prepared so that the proportion of blood in the reaction solution is 8%, and various PCNA mutants are added at 0.5 μg, 1 μg, 2 μg, and 4 μg to perform PCR reaction. The results of electrophoresis of the product obtained are shown. The PCNA mutants used are M73L and M73L / R82A / E143A. 0.5, 1, 2, and 4 of each photograph show the amount (μg) of added PCNA.
As a result, no PCNA was added, and even when 4 μg of the M73L mutant was added, amplification was not confirmed directly from the blood. However, when 0.5 μg of the M73L / R82A / E143A mutant was added, 8 μg of blood was firmly contained. Band was confirmed (FIG. 2).

It was confirmed that KOD-PCNA R109A / E143A mutant and Pfu-PCNA M73L / R109A / D143A and M73L / D143A / R82A mutants were also added to the same reaction as described above to improve the amplification amount.

(Example 6)
Production of KOD DNA polymerase mutant In order to be used for the evaluation of PCNA in Examples described later, a KOD DNA polymerase V93K mutant (hereinafter referred to as KOD V93K) was produced by the following method.
The plasmid of KOD V93K was prepared by inserting a mutation into a modified thermostable DNA polymerase gene (SEQ ID NO: 7) (pKOD) derived from Thermococcus kodakaraensis KOD1 strain cloned into pBlueScript.
The mutation was introduced by using KOD-Plus-Mutageness Kit (manufactured by Toyobo) according to the instruction manual. The mutant was confirmed by decoding the base sequence. Escherichia coli JM109 was transformed with the obtained plasmid and used for enzyme preparation.

Production of modified thermostable DNA polymerase The cells obtained above were cultured as follows. First, 80 mL of TB medium (Molecular cloning 2nd edition, p.A.2) containing sterilized 100 μg / mL ampicillin was dispensed into a 500 mL Sakaguchi flask. Escherichia cultivated for 16 hours at 37 ° C. in 3 mL of LB medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride; Gibco) containing 100 μg / mL ampicillin in advance in this medium. Coli JM109 (plasmid transformant) (using a test tube) was inoculated and cultured at 37 ° C. for 16 hours with aeration. The cells are collected from the culture solution by centrifugation, suspended in 50 mL of disruption buffer (30 mM Tris-HCl buffer (pH 8.0), 30 mM NaCl, 0.1 mM EDTA), and then subjected to sonication. By crushing, a cell lysate was obtained. Next, the cell lysate was treated at 80 ° C. for 15 minutes, and then the insoluble fraction was removed by centrifugation. Furthermore, nucleic acid treatment using polyethyleneimine, ammonium sulfate precipitation, and heparin sepharose chromatography were performed. Finally, storage buffer (50 mM Tris-HCl buffer (pH 8.0), 50 mM potassium chloride, 1 mM dithiothreitol, 0 1% Tween 20, 0.1% nonidet P40, 50% glycerin) to obtain a modified thermostable DNA polymerase.

The activity of the DNA polymerase in the purification step was measured as follows. If the enzyme activity is strong, the sample is stored in a storage buffer (50 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM dithiothreitol, 0.1% Tween 20, 0.1% Nonidet P40, 50% glycerin). Dilute and measure. (1) 25 μl of the following solution A, 5 μl of solution B, 5 μl of solution C, 10 μl of sterilized water, and 5 μl of enzyme solution are added to a microtube and reacted at 75 ° C. for 10 minutes. (2) Then, ice-cool, add 50 μl of E solution and 100 μl of D solution, and stir on ice for 10 minutes after stirring. (3) This solution is filtered through a glass filter (GF / C filter manufactured by Whatman) and thoroughly washed with 0.1N hydrochloric acid and ethanol. (4) The radioactivity of the filter is measured with a liquid scintillation counter (manufactured by Packard), and the incorporation of nucleotides into the template DNA is measured. One unit of enzyme activity is defined as the amount of enzyme that takes 10 nmol nucleotides per 30 minutes into the acid-insoluble fraction (that is, the fraction insolubilized when solution D is added) under these conditions.
A: 40 mM Tris-HCl buffer (pH 7.5) 16 mM magnesium chloride 15
mM dithiothreitol 100 μg / mL BSA (bovine serum albumin)
B: 1.5 μg / μl activated calf thymus DNA
C: 1.5 mM dNTP (250 cpm / pmol [3H] dTTP)
D: 20% trichloroacetic acid (2 mM sodium pyrophosphate)
E: 1 mg / mL calf thymus DNA

(Example 7)
Amplification in the presence of dUTP by adding PCNA Effect of adding KOD-PCNA mutants (M73L, M73L / R109A / E143A, M73L / R82A / E143A) in the presence of dUTP using KOD V93K obtained above. Compared.
For PCR, KOD-Plus-Ver. 2 (manufactured by Toyobo) 10 × PCR Buffer attached, 1 × PCR Buffer and 1.5 mM MgSO ₄ , 0.2 mM dNTPs containing dUTP instead of dTTP (dATP, dUTP, dCTP, dGTP), about 250 ng of PCNA to be evaluated was added to 50 μl of a reaction solution containing 15 pmol of the primer according to SEQ ID NOS: 8 and 9 for amplifying 1.3 kb, 10 ng of human genomic DNA (Roche), and 1U KOD DNA polymerase V93K mutant. After the pre-reaction at 94 ° C. for 30 seconds, PCR was performed on a schedule of 98 ° C., 10 seconds → 65 ° C., 30 seconds → 68 ° C., 1 minute 30 seconds repeated 35 cycles.
After completion of the reaction, 5 μl of the reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amplification amount of the amplified DNA fragment was confirmed under ultraviolet irradiation.

FIG. 3 shows the results of electrophoresis of the products obtained by adding 250 ng of various PCNA mutants and performing PCR reaction. The PCNA mutant used was a total of three types: M73L, M73L / R109A / E143A, and M73L / R82A / E143A.

The KOD V93K mutant used this time has a small amount of amplification due to dUTP inhibition. A band was hardly confirmed in the case of no PCNA addition or in the M73L mutant, but a firm band was confirmed by the addition of the KOD-PCNA mutant of M73L / R109A / E143A and M73L / R82A / E143A. PCNA forms a multimer and promotes a nucleic acid synthesis reaction. Usually, however, the reaction cannot proceed without loading into DNA without the action of RFC. The modification of R109A / E143A and R82A / E143A is a modification to the site involved in multimer formation. Since multimer formation was moderately weakened, PCNA could be loaded into DNA and the amount of PCR amplification could be improved. .

A similar experiment was performed with Pfu-PCNA mutants (M73L, M73L / R109A / D143A, M73L / R82A / D143A), and almost no band was confirmed with the Pfu-PCNA M73L mutant, but M73L / R109A / D143A, A firm band was confirmed by the addition of the M73L / R82A / D143A Pfu-PCNA mutant.

(Example 8)
Comparison of DNA polymerase synthesis rate by addition of PCNA The KOD-PCNA variant of M73L / R109A / E143A was added to the KOD V93K variant, and the synthesis rate was measured. KOD V93K was diluted with a storage buffer (50 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM dithiothreitol, 0.1% Tween 20, 0.1% Nonidet P40, 50% glycerin). In addition, 250 ng of PCNA mutant was added to each reaction system, and the synthesis rate was measured.
Reagents and methods were carried out according to the following <Method for measuring DNA polymerase synthesis rate>.

<Method for measuring DNA polymerase synthesis rate>
(1) Mix 2.5 μl of the following A liquid, 2.5 μl of B liquid, 1.5 μl of C liquid, 4.8 μl of D liquid, and 4 μl of E liquid, 10 minutes at 95 ° C., 10 minutes at 37 ° C. Place and create substrate.
(2) Add 5 μl of a 0.64 ng / μl enzyme solution to the substrate incubated at 75 ° C. for 30 seconds, and react at 75 ° C. for 30 seconds, 60 seconds, and 120 seconds. Then, ice-cool, add 35 μl of solution F, and stir well.
(3) This solution is applied to 10 μl, 1% alkaline agarose gel, and electrophoresed using Biotinylated 2-Log DNA Ladder (0.1-10 kb) (manufactured by NEB) as a marker.
(4) Blotting to Hybond N + and detecting biotin according to NEB's Phototope-Star Chemiluminescent Detection Kit manual. The length of the chain extended per second is defined as the synthesis rate.
A: 10 × PCR Buffer for KOD-Plus-Ver. 2 (TOYOBO)
B: 2 mM dNTPs (manufactured by TOYOBO)
C: 25 mM MgSO ₄
D: 200 ng / μl M13 ssDNA (manufactured by TaKaRa)
E: 100 μM Biotinylated P7 primer (Sequence: Biotin-CGCCAGGGTTTCCCAGTCACGAC)
F: 59 mM NaOH, 59 mM EDTA, 0.1% BPB 30% Glycerol

FIG. 4 shows the results obtained by adding the KOD-PCNA mutant, M73L / R109A / E143A to the V93K mutant, performing PCR reaction for 30 seconds, 60 seconds, and 120 seconds, and electrophoresing the resulting products. In FIG. 4, “30” indicates that the PCR reaction was performed for 30 seconds. The same applies to 60 and 120. FIG. 4 shows that the longer the amplified product is detected, the faster the synthesis rate, as the amplified product is detected at the top of the electrophoresis photograph. On the left side of the photograph, 7000 bp and 3000 bp amplification products are detected at the respective arrows. The numbers shown at the bottom of the photograph are the synthesis rates of each DNA polymerase calculated based on the results.

As a result, it was confirmed that when PCNA was added, the synthesis rate was significantly increased as compared to the case where PCNA was not added (left photo in FIG. 4) (FIG. 4). This is thought to be due to the fact that PCNA performed the function of clamping, and DNA and polymerase were bound firmly.

The present invention is useful in the biotechnology-related industry, and particularly useful in the technology related to DNA synthesis.

Claims (11)

Following (a) or (b) of the amino acid substitutions represented by Ri polypeptide Tona having at least one, and, PCNA monomer derived from the Pyrococcus (Pyrococcus) genus or Thermococcus (Thermococcus) genus of bacteria.
In the amino acid sequence described in (a) SEQ ID NO: 1, arginine corresponding to the 109th is substituted with a neutral amino acid, and polypeptide glutamate corresponding to 143 th has been substituted with a neutral amino acid (b) SEQ ID NO: 1. A polypeptide in which the arginine corresponding to the 82nd position is substituted with a neutral amino acid and the glutamic acid corresponding to the 143rd position is replaced with a neutral amino acid in the amino acid sequence described in 1. The PCNA monomer according to claim 1, wherein the neutral amino acid in (a) and (b) is alanine. The PCNA monomer according to claim 1 or 2, wherein, in the amino acid substitution represented by (a) or (b), the 73rd methionine is further substituted with leucine. DNA which codes the amino acid sequence with which the PCNA monomer in any one of Claims 1-3 is provided. A vector incorporating the DNA according to claim 4 . A transformant comprising the vector according to claim 5 . Culturing in a medium a transformant according to claim 6, wherein in the transformants or the culture medium, to accumulate multimer composed of PCNA monomer and / or the monomer, accumulated the PCNA A method for producing PCNA, comprising a step of recovering a monomer or a multimer. PCNA monomer and / or the DNA replication reagent comprising a multimer composed of monomers of any of claims 1-3. A DNA replication kit comprising the reagent according to claim 8 . A multimer composed of PCNA monomer and / or the monomers according to any one of claims 1-3, performed DNA synthesis reaction in the presence of a DNA polymerase replication process of DNA. The DNA replication method according to claim 9 , wherein the DNA synthesis reaction is PCR.