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WO2008066350A1 - A novel ditpase and genes encoding it - Google Patents

A novel ditpase and genes encoding it Download PDF

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Publication number
WO2008066350A1
WO2008066350A1 PCT/KR2007/006150 KR2007006150W WO2008066350A1 WO 2008066350 A1 WO2008066350 A1 WO 2008066350A1 KR 2007006150 W KR2007006150 W KR 2007006150W WO 2008066350 A1 WO2008066350 A1 WO 2008066350A1
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Prior art keywords
protein
dna
dna polymerase
ditpase
ditp
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PCT/KR2007/006150
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French (fr)
Inventor
Jung Hyun Lee
Sung Gyun Kang
Sang Jin Kim
Kae Kyoung Kwon
Hyun Sook Lee
Yun Jae Kim
Yong Gu Ryu
Seung Seob Bae
Jae Kyu Lim
Jung Ho Jeon
Yo Na Cho
Insoon Jeong
Suk Tae Kwon
Sun Shin Cha
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Korea Ocean Research & Development Institute
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Publication of WO2008066350A1 publication Critical patent/WO2008066350A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria

Definitions

  • the present invention relates to a novel dITPase, protein for enhancing DNA polymerase activity, and genes encoding it.
  • thermostable DNA polymerase which uses the thermostable DNA polymerase, is one of the most important contributions to protein and genetic research and is current Ly used in a broad array of biological applications. More than 50 DNA polymerase genes have been cloned from various organisms, including thermophiles and archaeas . Recently, family B DNA polymerases from hyperthermophilic archaea, Pyrococcus and Thermococcus, have been widely used since they have higher fidelity in PCR based on their proof reading activity than Taq polymerase commonly used. However, the improvement of the high fidelity enzyme has been on demand due to lower DNA elongation ability. [Background Art]
  • the present inventors isolated a new hyperthermophilic strain from a deep-sea hydrothermal vent area at the PACMANUS field. It was identified as a member of Thermococcus based on 16S rDNA sequence analysis, and the whole genome sequencing is currently in process to search for many extremely thermostable enzymes. The analysis of the genome information displayed that the strain possessed a family B type DNA polymerase. The present inventors cloned the gene corresponding to the DNA polymerase and expressed in E. coli. In addition, the recombinant enzyme was purified and its enzymatic characteristics were examined. Therefore, the present inventors applied for a patent on the DNA polymerase having high DNA elongation and high fidelity ability (Korean Patent Application No.
  • the present inventors isolated a HAM-I like protein from Thermococcus sp. and identified as a novel dITPase enhancing DNA polymerase proce ⁇ ssivity when it is used with DNA polymerase together.
  • the HAM-I like protein can be used for all of reactions of DNA polymerization.
  • the present inventors found that the protein according to the present invention can be used for reactions of DNA polymerization which is demanded for high sensitivity £ind rapidity, because it has a property of supporting DNA polymerase activity, thereby completing the present invention.
  • the present invention provides a dITPase protein and their functional equivalents. More specifically, the present invention provides dITPase protein and their functional equivalents, wherein the protein has the activity of enhancing DNA polymerase processivity.
  • the dITPase protein according to the present invention is the protein of SEQ ID NO: 1, and specifically, "functional equivalent" includes a protein for enhancing DNA polymerase activity having amino acids sequence shown in SEQ ID NO: 1, or amino acid sequence variants having amino acid substitutions in some or all of the protein for enhancing DNA polymerase, or amino acid additions or deletions in some of the protein for enhancing DNA polymerase.
  • the present invention includes the protein synthesized in vitro from public amino acids sequence .
  • the present invention provides a nucleotide sequence encoding the protein of SEQ ID NO: 1.
  • the nucleotide sequence is SEQ ID NO: 2.
  • nucleotide sequences encoding the protein of SEQ ID NO: 1 can be prepared diversely.
  • the present invention provides a recombinant vector comprising the gene of SEQ ID NO: 2.
  • vector means a nucleic acid molecule that can carry another nucleic acid bound thereto.
  • expression vector is intended to include a plasmid, cosmid or phage, which can synthesize a protein encoded by a recombinant gene carried by said vector.
  • a preferred vector is a vector that can self-replicate and express a nucleic acid bound thereto.
  • the present invention provides a host cell transformed with said recombinant vector.
  • transformation means that foreign DNA or RNA is absorbed into cells to change the genotype of the cells.
  • the present invention provides a method for producing the protein of SEQ ID NO: 1 by using said host cell.
  • Optional recombinant proteins for expression include human proteins, and mean all proteins to use for treating diseases, or to be applicable industrially.
  • the present invention provides a specific antibody against the protein of SEQ ID NO: 1, wherein the specific antibody is prepared by injecting the protein of SEQ ID NO: 1.
  • the present invention provides a method for enhancing DNA polymerization with the DNA polymerase by using said protein.
  • DNA polymerase refers to an enzyme that synthesizes DNA in the 5' -> 3' direction from deoxynucleotide triphosphate using a complementary template DNA strand and a primer by successively adding nucleotide to a free 3'-hydroxyl group.
  • the template strand determines the sequence of the added nucleotide by Watson- Crick base pairing.
  • PCR Polymerase Chain Reaction
  • a target DNA can be amplified more effectively.
  • the mixing of the protein of SEQ ID NO: 1 and a DNA polymerase can be simultaneously or differentially. That is one method is that mixture of the protein of SEQ ID NO: 1 and a DNA polymerase are added to PCR reaction solution, and then PCR reaction can be performed by adding target DNA fragment, and one another method is that target DNA are added to premix solution including a DNA polymerase, and then PCR reaction can be performed by adding the protein of SEQ ID NO: 1. [Advantageous Effects]
  • the dITPase, a HAM-I like protein, according to the present invention enhances DNA polymerase processivity when it is used with DNA polymerase together. Accordingly, the HAM-I like protein can be used for all reactions of DNA polymerization. Especially, the dITPase according to the present invention can be used for reactions of DNA polymerization which was demanded for high sensitivity and rapidity, because it has a property of supporting DNA polymerase activity. In this case, we identified the PCR reaction with dITPase and dUTPase together is more efficient (Fig. 12) .
  • FIG. 1 shows sequence alignments of TNAl HAM-I with other HAM-I like genes and dNTPase pyrophosphatase.
  • FIG. 2 shows the results of SDS-PAGE analysis of the recombinant Hisg-tagged TNAl HAM-I.
  • Lane 1 low molecular range standard (Bio-Rad) ;
  • Lane 2 TNAl HAM-I purified by His-tagged affinity chromatography.
  • FIG. 3 shows effect of dITP on PCR amplification with various DNA polymerases.
  • A PCR amplification by rTaq and TNAl DNA polymerase with various concentrations of dITP (0, 0.025, 0.05, 0.1, 0.2mM);
  • B PCR amplification by various DNA polymerases with dITP (0.05mM dITP) and without dlTP (dITP X) .
  • FIG. 4 shows effect of TNAl HAM-I on E 3 CR amplification with various DNA polymerases.
  • FIG. 5 shows a cleavage map of recombinant plasmid according to the present invention.
  • FIG. 6 shows effect of dITP on PCR amplification by family A and B type DNA polymerases .
  • a 2 kb target from ⁇ DNA was amplified using 2.5 units of rTaq (Takara) or ].5 units of various family B-type DNA polymerases .
  • the reaction mixture contain 5 ng of ⁇ DNA, 10 pmol of primers, 0.25 UiM dNTPs, 0.05 mM dITP, and PCR reaction buffer.
  • FIG. 7 shows multi-alignment of the uracil-sensing domain of archaeal family B-type DNA polymerases from Pyrococcus and Thermococcus genus .
  • the amino acid sequence accession numbers are Pyrococcus horikoshii (059610) , P. abyssi (P77916) , P. glycovorans (CAC12849) , Pyrococcus sp. GE23 (CAA90887), Pyrococcus sp. GB-D (Q51334), P. furiosus (P61875) , P. woesei (P61876) , Thermococcus kodakaraensis
  • FIG. 8 shows primer extension assays using various archaeal family B-type DNA polymerases . Templates containing a single uracil (A) , or two successive uracils (B) were used. The first and second lanes in each panel exhibit 23-mer primer and 44-mer template, respectively.
  • FIG. 9 shows primer extension assays using TNAl DNA polymerase in the presence of hypoxanthine .
  • Templates containing no deaminated base (A) , two successive uracils (B) , or two successive hypoxanthines (C) were used.
  • the first and second lanes exhibit 23-mer primer and 44-mer template, respectively.
  • D PCR amplification using wild- type and mutant of TNAl DNA polymerases in the presence of dITP.
  • the reaction mixture contained 5 ng of ⁇ DNA, 10 pmol of primers, 0.25 mM dNTPs, 0.025 mM dITP, and PCR reaction buffer.
  • FIG. 10 shows whole domain swapping experiments.
  • A Schematic representation of Pfu and TNAl fusion proteins. The numbers indicate the residues of NAl.
  • B and C primer extension assays using Pfu and TNAl fusion proteins show the recognition of uracil. Templates containing a single uracil (B) or two successive uracils (C) were used. The first lane shows 23-mer primer and 44-mer template, respectively.
  • FIG. 11 shows primer extension assays using Pfu, TNAl, and KODl DNA polymerases. Templates containing a single uracil were used. The first and second lanes exhibit 23-mer primer and 44-mer template, respectively.
  • FIG. 12 shows effect of dITPase on PCR amplification. PCR was performed with TNAl (A) or Pfu DNA polymerases (B) in the presence of dITPase and dUTPase. The reaction mixture contained 5 ng of ⁇ DNA, 10 pmol of primers, 0.35 mM dNTPs, 5 ng dITPase or dUTPase, and PCR reaction buffer.
  • FIG. 13 shows dITP incorporation assay using PEu, KODl and TNAl family B-type DNA polymerases.
  • the reaction mixture contained primer-template complexs (200 fmol), 1.25 units of TNAl, 50 mM Tris-HCl pH 8.5, 60 mM KCl, 30 mM (NH 4 ) 2 SO 4 , 1 mM MgCl 2 , and only 0.1 mM dITP.
  • FIG. 14 shows primer extension assays using Pfu DNA polymerase.
  • FIG. 15 shows superimposed uracil-sensing domains and molecular docking of hypoxanthine. C ⁇ -tracing of superposed uracil-sensing domains of 0.313 A RSMD from KODl DNA polymerase (red; PDB code, IWNS) and Pfu DNA polymerase
  • FIG. 16 shows primer extension assays using various archaeal family B-type DNA polymerases .
  • Templates containing no deaminated base (A) , a single xanthine (B) , two successive xanthines (C) and four successive xanthines (D) were used.
  • the first and second lanes in each panel exhibit 23-mer primer and 44-mer template, respectively.
  • FIG. 17 shows optimal conditions of the nucleotide hydrolysis activities of dITPase.
  • FIG. 18 shows dynamic effect on hydrolysis of nucleotide triphosphate by dITPase and dUTPase.
  • the present invention provides a dITPase protein and their functional equivalents . More specifically, the present invention provides dITPase protein and their functional equivalents having the activity of enhancing DNA polymerase processivity.
  • the dITPase protein according to the present invention is the protein of SEQ ID NO: 1, and specifically, "functional equivalent" includes a protein for enhancing DNA polymerase activity having amino acids sequence shown in SEQ ID NO: 1, or amino acid sequence variants having amino acid substitutions in some or all of the protein for enhancing DNA polymerase, or amino acid additions or deletions in some of the protein for enhancing DNA polymerase.
  • the present invention includes the protein synthesized in vitro from public amino acids sequence.
  • the present invention includes the purified protein comprising amino acids sequence with greater than 55% similarity to amino acids sequence shown in SEQ ID NO: 1.
  • the peptides have greater than 60% sequence similarity. More preferably, the peptides have greater than 70% sequence similarity. Most preferably, the peptides have greater than 80% sequence similarity.
  • amino acids sequence with more than 55% similarity means at least 55% identical or conservatively replaced amino acid residues in a like position when aligned optimally allowing for up to 4 gaps with the proviso that in respect of each gap a total not more than 10 amino acid residues is affected.
  • the amino acid substitutions are preferably conservative substitutions.
  • conservative substitutions of naturally occurring amino acids include aliphatic amino acids (GIy, Ala, and Pro) , hydrophobic amino acids (lie, Leu, and VaI) , aromatic amino acids (Phe, Tyr, and Trp) , acidic amino acids (Asp, and GIu) , basic amino acids (His, Lys, Arg, GIn, and Asn) , and sulfur- containing amino acids (Cys, and Met) .
  • the deletions of amino acids are located in a region which is not involved directly in enhancing the activity of DNA polymerization.
  • the present invention provides a nucleotide sequence encoding the protein of SEQ ID NO: 1. Specifically, the nucleotide sequence is SEQ ID NO: 2.
  • nucleotide sequences encoding the protein of SEQ ID NO: 1 can be prepared diversely.
  • nucleic acid sequence is intended to include natural mRNA identified selective expression, complementary DNA (cDNA) sequence and equivalent nucleic acid sequence thereof.
  • equivalent nucleic acid sequence is intended to include sequences provided herein, sequences with allelic variation and with variation between species, or degenerate codon sequence.
  • the term “degenerate codon sequence” refers to a nucleic acid sequence, which is different from said naturally occurring sequence, but encodes a polypeptide having the same sequence as that of the polypeptide of SEQ ID NO: 1 disclosed in the present invention.
  • sequences with allelic variation or “a sequence wit h variation between species” refers to a nucleic acid sequence, which is different from said naturally occurring nucleic acid sequence, but encodes a polypeptide having practically the same functional feature as that of said polypeptide disclosed herein.
  • the present invention provides a recombinant vector comprising the gene of SEQ ID NO: 2.
  • vector means a nucleic acid molecule that can carry another nucleic acid bound thereto.
  • expression vector is intended to include a plasmid, cosmid or phage, which can synthesize a protein encoded by a recombinant gene carried by said vector.
  • a preferred vector is a vector that can self-replicate and express a nucleic acid bound thereto.
  • the present invention provides a host cell transformed with said recombinant vector.
  • transformation means that foreign DNA or RNA is absorbed into cells to change the genotype of the cells.
  • Host cells suitable for transformation include prokaryotic, fungal, plant and animal cells, but are not limited thereto. Most preferably, E. coli cells are used. Methods for culturing E. coli are well known in the art.
  • the present invention provides a method for producing the protein of SEQ ID NO: 1 using said host cell.
  • Optional recombinant proteins for expression include human proteins, and mean all proteins to use for treating diseases, or to be applicable industrially.
  • the present invention provides a specific antibody against the protein of SEQ ID NO: 1, wherein the specific antibody is prepared by injecting the protein of SEQ ID NO: 1.
  • the present invention provides a method for enhancing DNA polymerization with the DNA polymerase by using said protein.
  • DNA polymerase refers to an enzyme that synthesizes DNA m the 5' -> 3' direction from deoxynucleotide triphosphate using a complementary template DNA strand and a primer by successively adding nucleotide to a free 3'-hydroxyl group.
  • the template strand determines the sequence of the added nucleotide by Watson-Crick base pairing.
  • a target DNA can be amplified more efficiently.
  • the method of mixing of the protein of SEQ ID NO: 1 and a DNA polymerase can be simultaneously or differentially. That is one method is that mixture of the protein of SEQ ID NO: 1 and a DNA polymerase are added to PCR reaction solution, and then PCR reaction can be performed by adding target DNA fragment, and one another method is that target DNA are added to premix solution including a DNA polymerase, and then PCR reaction can be performed by adding the protein of SEQ ID NO: 1.
  • Thermococcus sp. NAl was isolated from a deep-sea hydrothermal vent area in the East Manus Basin. YPS medium was used to culture Thermococcus sp. NAl for DNA manipulation. Culture and strain maintenance were performed according to standard procedures. To prepare a seed culture of Thermococcus sp. NAl, YPS medium in a 25-ml serum bottle was inoculated with a single colony from a phytagel plate and cultured at 90 0 C for 20 h. Seed cultures were used to inoculate 700 ml of YPS medium in an anaerobic jar and cultured at 90°C for 2O h. E.
  • E. coli strain DH5 ⁇ was used for plasmid propagation and nucleotide sequencing.
  • E. coli strain BL21-CodonPlus (DE3) -RIL cells (Stratagene, LaJoIIa, CA) and the plasmid pET-24a(+) (Novagen, Madison, WI) were used for gene expression.
  • E. coli strains were cultivated in Luria-Bertani medium with 50 ⁇ g/ml kanamycin at 37°C.
  • DNA manipulation and sequencing DNA manipulations were performed using standard procedures, as described by Sambrook and Russell [Sambrook, J. & Russell, D. W., Molecular cloning: a laboratory manual, 3 rd ed., Cold Spring Harbor, N. Y. (2001)]. Genomic DNA of Thermococcus sp. NAl was isolated using a standard procedure. Restriction enzymes and other modifying enzymes were purchased from Promega (Madison, WI) . Small-scale preparation of plasmid DNA from E. coli cells was performed
  • DNA sequencing was performed using an ABI3100 automated sequencer, using a BigDye terminator kit (PE Applied Biosystems, Foster City, CA) .
  • TNAl HAM-I encoding gene The full length of Thermococcus sp. NAl HAM-I gene flanked by Ndel and Xhol sites was amplified by PCR using two primers and genomic DNA as a template.
  • the amplified fragment was digested with Ndel and Xhol, and ligated with pET-24a(+) digested with Ndel/Xhol.
  • the ligate was transformed into E. coli DH5 ⁇ .
  • the resulting plasmid was transformed into E. coli BL21-CodonPlus (DE3) - RIL and E. coli Rosetta (DE3) pLysS strain, respectively.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • the cells were harvested by centrifugation (6000 x g at 4 ° C for 20 min) and resuspended in 50 mM Tris-HCl buffer (pH 8.0) containing 0.1 M KCl and 10% glycerol. The cells were disrupted by sonication and harvested by centrifugation (20,000 x g at 4 ° C for 30 min). The resulting supernatant
  • the concentrations of proteins were determined by the colorimetric assay of Bradford (1976) .
  • the purification degrees of the proteins were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis according to a standard method (Laemmli, 1970) .
  • the TNAl HAM-I gene amplified by PCR was cloned into pET-24a(+) and transformed into E. coli BL21- CodonPlus (DE3)-RIL, and then expressed.
  • E. coli BL21- CodonPlus (DE3)-RIL
  • SDS-PAGE analysis the 95% purified enzyme revealed a single protein band with a molecular mass of 21 kDa, which was deduced amino acid sequence (FIG. 2).
  • TNAl HAM-I protein and a high fidelity DNA polymerase.
  • 50 ng of TNAl HAM-I protein was added to PCR mixture with various high fidelity DNA polymerases, respectively.
  • PCR mixture was consisted of 2.5U of DNA polymerase, 150 ng of genomic DNA from Thermococcus sp. NAl as a template, 10 pmole of each primer, 200 ⁇ M dNTPs and PCR reaction buffer.
  • Thermococcus sp. NAl, primers were designed (Table 1).
  • dITP dITP was automatically generated by deamination of dATP on PCR amplification and considered to be mainly responsible for decreasing PCR efficiency.
  • effects of TNAl HAM-I in addition to PCR reaction with high fidelity DNA polymerase were examined.
  • PCR reaction with high fidelity DNA polymerase in presence of TNAl HAM-I protein could be amplified a target DNA more effectively than in absence of TNAl HAM-I protein. It was expected that elimination of dITP by TNAl HAM-I protein in PCR reaction solution would be more effective in PCR amplification due to inhibition of stalling .
  • Plasmids were transformed Into Escherichia coli BL21 Rosetta (DE3)pLysS. Overexpression of genes was induced by addition of isopropyl- ⁇ - D -thiogalactopyranoside (IPTG) at the mid-exponential growth phase, followed by additional 3 h incubation at 37 0 C. Cells were then, harvested by centrifugation (6000 x g at 4 °C for 20 min) , and resuspended in 50 mM Tris-HCl buffer (pH 8.0) containing 0.1 M KCl and 10% glycerol, followed by sonication.
  • IPTG isopropyl- ⁇ - D -thiogalactopyranoside
  • the cell lysate was centrifuged (20,000 x g at 4 0 C for 30 min), and crude samples were prepared by heat treatment at 80 0 C for 20 min.
  • the resulting supernatant was applied to a column of TALON metal affinity resin (BD Biosciences, Clontech, Palo Alto, CA) and washed with 10 mM imidazole (Sigma, St. Louis, MO) in 50 mM Tris-HCl buffer (pH 8.0) containing 0.1 M KCl and 10% glycerol, and the protein was eluted with 300 mM imidazole in the same buffer.
  • the pooled fractions were dialyzed in storage buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM DTT, 1 mM EDTA, and 10% glycerol.
  • Primer extension assay was performed as described by Fogg et al. with slight modification (Fogg MJ, Pearl LH, Connolly BA, 2002, Nature Struct Biol 9:922-927). In brief, templates (Table 3) containing uracil, hypoxanthine or xanthine were used.
  • the primer-template complex (400 fmol) was incubated at 75 0 C for 15 min with 1.25 units of TNAl DNA polymerase in 120 mM Tris-HCl (pH 8.8), 60 inM KCl, 30 mM (NH 4 ) 2 SO 4 , 1 mM MgCl 2 , and 0.25 mM of each dNTP.
  • the reaction mixtures for rTaq (Takara) , Pfu (Promega) , Deep-vent (New England Biolabs, Inc., Beverly, Mass), Vent (New England Biolabs, Inc., Beverly, Mass), and KODl (Novagen) DNA polymerases were slightly modified using each manufacturer's buffer. After incubation, each sample was analyzed by 15% polyacrylamide gel electrophoresis followed by autoradiography.
  • TNAl DNA polymerase buffer consisted of 120 mM Tris-HCl (pH 8.8), 30 mM (NH 4 J 2 SO 4 , 60 mM KCl, and 1 mM MgCl 2 After single 1 min denaturation step at 95 °C, 30 cycles of denaturation (20 sec at 94 0 C) , annealing and extension (2 kb, 2 min; 5 kb, 3.5 min; 8 kb, 5 min; 10 kb,
  • uracil-sensing domain For the structural analysis of uracil-sensing domain, PDB ID, 2JGU and IWNS were used. A hypoxantine was snugly docked into into the binding pocket in the uracil-sensing domain using the "solid docking" module in QUANTA (Molecular Simulation, Inc.). The program considers an electrostatic and geometric complementarity in docking a guest molecule into a host molecule.
  • dITP inhibits PCR amplification of family B-type DNA polymerases It was found that PCR amplification of family B-type DNA polymerases from hyperthermophilic archaea was significantly inhibited by dITP whereas Taq, a family A- type DNA polymerase, was not (FIG. 6) . Biochemical and structural investigation of Pfu DNA polymerase to unveil the molecular basis for the inhibitory effect of dITP demonstrated that dITP was incorporated into newly synthesized strands in the iirst round of the PCR, and subsequently uracil-sensing domain would sense the incorporated hypoxanthine in the template, eventually stalling replication (FIG. 13, FIG. 14 and FIG. 15) .
  • Pfu, KODl and TNAl polymerases could replicate the template, indicating that the polymerase activity of Pfu, KODl and TNAl polymerases was not inhibited by dITP (FIG. 13) .
  • dITP dITP
  • Pfu DNA polymerase stopped right after adding a hypoxanthine to pair with cytosine. It is known that the paring with cytosine is the most stable even though hypoxanthine can pair with all four natural bases.
  • KODl and TNAl DNA polymerases could replicate the template up to the end (44-mer) using only dITP as a sole dNTP. It was thought that KODl and TNAl DNA polymerases were relatively insensitive to the wobble base pairing between hypoxanthine and other bases while Pfu DNA polymerase extremely prefers hypoxanthine and cytosine base-paring. Regardless of the disparity in the incorporation pattern, it was obvious that DNA polymerase activity itself was not affected by the presence of dITP.
  • primer extension assay was performed to test whether the presence of hypoxanthine in the DNA template was a cause for the inhibitory effect of dITP.
  • Pfu DNA polymerase was stalled at a DNA template with hypoxanthine, similar to stalling at uracil, raising the possibility that Pfu DNA polymerase can sense hypoxanthine in the DNA template.
  • structural and biochemical analysis was performed as followed.
  • uracil-sensing domain of the DNA polymerase was responsible for recognizing hypoxanthine in that mutations at critical residues (Y7, V93, and F116) in uracil-sensing domain could get over the stalling in the presence of hypoxanthine (FIG. 14), consistent with the recent report (Gill S. et al . , 2007, J MoI Biol) .
  • Molecular docking model supports the finding that hypoxanthine was well fitted to uracil sensing domain (FIG. 15) .
  • the docking with uracil may be the most favorable than other bases with less free energy, however, the hole could hold hypoxanthine as well .
  • dITP seemed to be incorporated in the first round of PCR, and then sensed by uracil-sensing domain, stalling DNA polymerases at the encountering hypoxanthine. Consistent with the observation with, family B-type DNA polymerases were also stalled in the presence of xanthine. However, the stalling position at xanthine was different (FIG. 16) .
  • chimeric proteins generated by whole domain swapping of uracil-sensing domains or exonuclease domains between Pfu and TNAl DNA polymerases exhibited similar stalling patterns to the corresponding wild type proteins.
  • a fusion protein with the uracil-sensing domain from Pfu DNA polymerase and the exonuclease/polymerase domain from NAl followed the pattern of TNAl DNA polymerase and vice versa (FIG. 10) . Consequently, it is reasonable to conclude that the discrepancy in the stalling pattern was neither brought about by uracil-sensing domain nor exonuclease domain.
  • TNAl and KODl DNA polymerases are superior to other DNA polymerases in extension rates.
  • TNAl and KODl DNA polymerase has three times and six times higher extension rate than Pfu and Deep-Vent DNA polymerases, respectively.
  • TNAl and KODl DNA polymerases were stalled at as low as 45 °C by a single deaminated base, but the stalling disappeared at higher temperatures (FIG. 11) .
  • TNAl DNA polymerase with three times higher extension rate than the wild type protein.
  • two successive deaminated bases were not enough to stall the mutant protein.
  • the result converges to support the close correlation between the stalling pattern and the extension rate.
  • the reason why the binding of the first deaminated base to uracil-sensing domain fails to stall TNAl and KODl polymerases would be that the binding energy was not enough to stall the fast- moving enzyme.
  • the interaction between the first deaminate base and the uracil-sensing domain could play as a brake to retard the enzyme, and subsequently, the DNA polymerase with a decreased mobility is then stalled by the second deaminated base.
  • This stalling mechanism of DNA polymerases may explain why DNA polymerases with higher extension rate require more deaminated bases to be stalled.
  • the binding of uracil (hypoxantine) to uracil- sensing domain which commonly occurs in family B-type DNA polymerases, does not necessarily lead to the stalling of enzymes. The stalling is highly likely to be determined by both the extension rate of DNA polymerase and the recognition of deaminated bases .
  • DNA polymerase works in concert with various auxiliary factors such as PCNA, replication factor, helicase and so on. It is possible that the factors or the condition in vivo could change the micro-environment of the enzyme, enable the DNA polymerases to sense even one base.
  • PCR conditions are as follows: 0.10 mM dATP cycled alone or with 5.00 ng ⁇ l-1 native dITPase. - Twenty five cycles: 95 0 C for 1 min, 95 0 C for 20 s, 72 0 C for 8 min, 72 0 C for 7 min.
  • TNA1__HAM1 A HAMl-like protein homologue (TNA1__HAM1) from Thermococcus onnurineus NAl was cloned and expressed in E. coli. TNA1_HAM1 showed similarities to HAMl-like protein from Thermococcus sp.
  • the purified TNA1_HAM1 could eliminate the generated dITP during PCR amplification, and enhanced PCR amplification yield by TNAl and Pfu DNA polymerases (FIG. 12) .
  • the characterization of purified HAMIp Choung JH et al., 2001, Nucleic Acids Res 29:3099- 3107) and dUTPase (Hogrefe HH et al., 2001, Proc Natl Acad Sci USA 99:596-601., Cho Y et al., 2007, Mar Blotechnol (In press) ) showed that the two enzymes are very specific to hypoxanthine/xanthine and uracil dNTP, respectively.
  • PCR yield by adding TNA1_HAM1 may be cumulative effects of hydrolyzing dITP and dXTP, considering that dGTP deamination could take place.
  • the recognition (sensing) of deaminated bases is an intrinsic property of family B-type DNA polymerases of hyperthermophilic archaea living at temperatures above 80 0 C, preventing transition mutation of deaminated bases.
  • the stalling of DNA polymerase seems to rely on the molecular mobility during DNA replication.
  • dITPase is a strategic, effective way to escape the inhibitory effect of dITP or dXTP in the PCR.
  • the effects of pH on the nucleotide hydrolysis activities of dITPase were analyzed under various pH conditions with the substrate dITP (FIG. 17) .
  • the nucleotide hydrolysis activity of dITPase has alkaline pH.
  • the reaction rates under neutraL conditions were ⁇ 10% of maximum (FIG. 17).
  • Dependence of the nucleotide hydrolysis activities of dITPase on temperature was also investigated.
  • the optimal reaction temperature of dITPase was 70 ° C, but the activities was greatly reduced above 90 ° C (FIG. 17) .
  • reaction for dUTPase was carried out in 50 ⁇ l of reaction mixture containing lxTris-HCl buffer (120 mM Tri-HCl, 1 mM
  • reaction mixture 50 ⁇ l contains 100 mM dATP and lxBuffer (120 mM Tris-HCl, 1 mM MgCl 2 , 60 mM KCl, 30 mM (NH 4 ) 2 SO 4 , and 0.01% BSA), and was analyzed after 12 kb- targeted PCR reaction. Thermal condition is same as PCR condition.
  • dITPase 5 ng was used as a same time to check its effect on removing by-product (dITP) .
  • the reaction products were chromatographed on Partisil-10 SAX column
  • the amount of dITP, which is produced during PCR reaction, has been carried out by HPLC.
  • the peak expected to dITP was detected at 7.7-7.9 min and it was removed when dITPase was added to PCR mixture. It was observed that 0.93% of dATP was converted to dITP during PCR reaction (Table 4) .
  • dITPase and dUTPase were added to amplifications of 2, 5, 8, 10, and 12 kb lambda DNA targets conducted with cloned TNAl DNA polymerase (20 ng) , using extension time (2, 3.5, 5, 6, and 7 min) . Enhancing activity was identified by a visible increase in product yield.
  • Two step reaction was used to amplify each DNA fragment under following conditions: an initial denaturation step of 1 min at 95 0 C, 25 cycles of amplification (20 s at 95°C, 2, 3.5, 5, 6, and 7 min at 72 0 C), and a final extension period of 5 min at 72 0 C.
  • Oxcidative deamination of DNA primarily convertes DNA base amino groups to keto groups. Thus, adenine is converted to hypoxanthine (HX) , guanine to xanthine (X) , and cytosine to uracil (U) . Also, a decrease in the activities of DNA polymerase, which is belonging to Family type B (TNAl, Pfu, and KOD) seemed to be slightly reduced by dITP and dUTP (Kim et al . , 2007, unpublished data). They are produced by deamination of DNA base amino group under high temperature condition followed by a decrease in product yield.
  • dITPase according to the present invention could be used for reactions of DNA amplification which was demanded for high sensitivity and rapidity, as supporting DNA polymerase activity.

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Abstract

The present invention relates to a novel dITPase, protein for enhancing DNA polymerase activity, and genes encoding it. More specifically, the present invention relates to a dITPase isolated from Thermococcus sp NAl. strain. Also, the present invention provides gene fragments encoding said protein, recombinant vectors containing thereof, host cells transformed with thereof and methods for producing dITPase using said host cells.

Description

[Description] [invention Title]
A NOVEL dITPase AND GENES ENCODING IT
[Technical Field] The present invention relates to a novel dITPase, protein for enhancing DNA polymerase activity, and genes encoding it.
The recent advance of genomic research has produced vast amounts of sequence information. With a generally applicable combination of conventional genetic engineering and genomic research techniques, the genome sequences of some hyperthermophilic microorganisms are of considerable biotechnological interest due to heat-stable enzymes, and many extremely thermostable enzymes are being developed for biotechnological purposes.
PCR, which uses the thermostable DNA polymerase, is one of the most important contributions to protein and genetic research and is current Ly used in a broad array of biological applications. More than 50 DNA polymerase genes have been cloned from various organisms, including thermophiles and archaeas . Recently, family B DNA polymerases from hyperthermophilic archaea, Pyrococcus and Thermococcus, have been widely used since they have higher fidelity in PCR based on their proof reading activity than Taq polymerase commonly used. However, the improvement of the high fidelity enzyme has been on demand due to lower DNA elongation ability. [Background Art]
The present inventors isolated a new hyperthermophilic strain from a deep-sea hydrothermal vent area at the PACMANUS field. It was identified as a member of Thermococcus based on 16S rDNA sequence analysis, and the whole genome sequencing is currently in process to search for many extremely thermostable enzymes. The analysis of the genome information displayed that the strain possessed a family B type DNA polymerase. The present inventors cloned the gene corresponding to the DNA polymerase and expressed in E. coli. In addition, the recombinant enzyme was purified and its enzymatic characteristics were examined. Therefore, the present inventors applied for a patent on the DNA polymerase having high DNA elongation and high fidelity ability (Korean Patent Application No. 2005-0094644) . But because of strong exonuclease activity and low processivity, high fidelity DNA polymerases need to improve in various applications of PCR. The present inventors isolated a HAM-I like protein from Thermococcus sp. and identified as a novel dITPase enhancing DNA polymerase proce^ssivity when it is used with DNA polymerase together. The HAM-I like protein can be used for all of reactions of DNA polymerization. Especially, the present inventors found that the protein according to the present invention can be used for reactions of DNA polymerization which is demanded for high sensitivity £ind rapidity, because it has a property of supporting DNA polymerase activity, thereby completing the present invention.
[Disclosure] [Technical Problem]
It is an object of the present invention to provide a novel protein for enhancing DNA polymerase activity, and genes encoding it. Also, another object of the present invention is to provide a recombinant vector prepared by inserting said genes, and a method for producing the protein for enhancing DNA polymerase activity using a host cell transformed with said recombinant vector. [Technical Solution]
To achieve the above aim, the present invention provides a dITPase protein and their functional equivalents. More specifically, the present invention provides dITPase protein and their functional equivalents, wherein the protein has the activity of enhancing DNA polymerase processivity. Preferably, the dITPase protein according to the present invention is the protein of SEQ ID NO: 1, and specifically, "functional equivalent" includes a protein for enhancing DNA polymerase activity having amino acids sequence shown in SEQ ID NO: 1, or amino acid sequence variants having amino acid substitutions in some or all of the protein for enhancing DNA polymerase, or amino acid additions or deletions in some of the protein for enhancing DNA polymerase. Also, the present invention includes the protein synthesized in vitro from public amino acids sequence .
The present invention provides a nucleotide sequence encoding the protein of SEQ ID NO: 1. Specifically, the nucleotide sequence is SEQ ID NO: 2. As a result of the degeneracy of the genetic code, nucleotide sequences encoding the protein of SEQ ID NO: 1 can be prepared diversely.
The present invention provides a recombinant vector comprising the gene of SEQ ID NO: 2. As used herein, the term "vector" means a nucleic acid molecule that can carry another nucleic acid bound thereto. As used herein, the term "expression vector" is intended to include a plasmid, cosmid or phage, which can synthesize a protein encoded by a recombinant gene carried by said vector. A preferred vector is a vector that can self-replicate and express a nucleic acid bound thereto.
The present invention provides a host cell transformed with said recombinant vector. As used herein, the term "transformation" means that foreign DNA or RNA is absorbed into cells to change the genotype of the cells.
The present invention provides a method for producing the protein of SEQ ID NO: 1 by using said host cell. Optional recombinant proteins for expression include human proteins, and mean all proteins to use for treating diseases, or to be applicable industrially. The present invention provides a specific antibody against the protein of SEQ ID NO: 1, wherein the specific antibody is prepared by injecting the protein of SEQ ID NO: 1.
The present invention provides a method for enhancing DNA polymerization with the DNA polymerase by using said protein. As used herein, the term "DNA polymerase" refers to an enzyme that synthesizes DNA in the 5' -> 3' direction from deoxynucleotide triphosphate using a complementary template DNA strand and a primer by successively adding nucleotide to a free 3'-hydroxyl group. The template strand determines the sequence of the added nucleotide by Watson- Crick base pairing.
With mixture of the protein of SEQ ID NO: 1 and a DNA polymerase, the rate of DNA replication has increased. Accordingly, with mixture of the protein of SEQ ID NO: 1 and a DNA polymerase in Polymerase Chain Reaction (PCR), a target DNA can be amplified more effectively. The mixing of the protein of SEQ ID NO: 1 and a DNA polymerase can be simultaneously or differentially. That is one method is that mixture of the protein of SEQ ID NO: 1 and a DNA polymerase are added to PCR reaction solution, and then PCR reaction can be performed by adding target DNA fragment, and one another method is that target DNA are added to premix solution including a DNA polymerase, and then PCR reaction can be performed by adding the protein of SEQ ID NO: 1. [Advantageous Effects]
The dITPase, a HAM-I like protein, according to the present invention enhances DNA polymerase processivity when it is used with DNA polymerase together. Accordingly, the HAM-I like protein can be used for all reactions of DNA polymerization. Especially, the dITPase according to the present invention can be used for reactions of DNA polymerization which was demanded for high sensitivity and rapidity, because it has a property of supporting DNA polymerase activity. In this case, we identified the PCR reaction with dITPase and dUTPase together is more efficient (Fig. 12) .
[Description of Drawings]
FIG. 1 shows sequence alignments of TNAl HAM-I with other HAM-I like genes and dNTPase pyrophosphatase.
FIG. 2 shows the results of SDS-PAGE analysis of the recombinant Hisg-tagged TNAl HAM-I. Lane 1, low molecular range standard (Bio-Rad) ; Lane 2, TNAl HAM-I purified by His-tagged affinity chromatography. FIG. 3 shows effect of dITP on PCR amplification with various DNA polymerases. A, PCR amplification by rTaq and TNAl DNA polymerase with various concentrations of dITP (0, 0.025, 0.05, 0.1, 0.2mM); B, PCR amplification by various DNA polymerases with dITP (0.05mM dITP) and without dlTP (dITP X) .
FIG. 4 shows effect of TNAl HAM-I on E3CR amplification with various DNA polymerases.
FIG. 5 shows a cleavage map of recombinant plasmid according to the present invention.
FIG. 6 shows effect of dITP on PCR amplification by family A and B type DNA polymerases . A 2 kb target from λ DNA was amplified using 2.5 units of rTaq (Takara) or ].5 units of various family B-type DNA polymerases . The reaction mixture contain 5 ng of λ DNA, 10 pmol of primers, 0.25 UiM dNTPs, 0.05 mM dITP, and PCR reaction buffer.
FIG. 7 shows multi-alignment of the uracil-sensing domain of archaeal family B-type DNA polymerases from Pyrococcus and Thermococcus genus . The amino acid sequence accession numbers are Pyrococcus horikoshii (059610) , P. abyssi (P77916) , P. glycovorans (CAC12849) , Pyrococcus sp. GE23 (CAA90887), Pyrococcus sp. GB-D (Q51334), P. furiosus (P61875) , P. woesei (P61876) , Thermococcus kodakaraensis
(accession no. P77933) , T. gorgonarius (P56689) , T. fumicolans (P74918), T. sp. 9°N-7 (Q56366) , T. onnurineus
NAl (ABC11972), T. litoralis (P30317) , and T. aggregάns
(033845) . FIG. 8 shows primer extension assays using various archaeal family B-type DNA polymerases . Templates containing a single uracil (A) , or two successive uracils (B) were used. The first and second lanes in each panel exhibit 23-mer primer and 44-mer template, respectively.
FIG. 9 shows primer extension assays using TNAl DNA polymerase in the presence of hypoxanthine . Templates containing no deaminated base (A) , two successive uracils (B) , or two successive hypoxanthines (C) were used. The first and second lanes exhibit 23-mer primer and 44-mer template, respectively. (D) , PCR amplification using wild- type and mutant of TNAl DNA polymerases in the presence of dITP. The reaction mixture contained 5 ng of λ DNA, 10 pmol of primers, 0.25 mM dNTPs, 0.025 mM dITP, and PCR reaction buffer.
FIG. 10 shows whole domain swapping experiments. (A), Schematic representation of Pfu and TNAl fusion proteins. The numbers indicate the residues of NAl. In (B) and (C), primer extension assays using Pfu and TNAl fusion proteins show the recognition of uracil. Templates containing a single uracil (B) or two successive uracils (C) were used. The first lane shows 23-mer primer and 44-mer template, respectively.
FIG. 11 shows primer extension assays using Pfu, TNAl, and KODl DNA polymerases. Templates containing a single uracil were used. The first and second lanes exhibit 23-mer primer and 44-mer template, respectively. FIG. 12 shows effect of dITPase on PCR amplification. PCR was performed with TNAl (A) or Pfu DNA polymerases (B) in the presence of dITPase and dUTPase. The reaction mixture contained 5 ng of λ DNA, 10 pmol of primers, 0.35 mM dNTPs, 5 ng dITPase or dUTPase, and PCR reaction buffer. (C) , SDS-PAGE analysis of purified dITPase from T. onnurineus NAl. Lane M, molecular weight standards. FIG. 13 shows dITP incorporation assay using PEu, KODl and TNAl family B-type DNA polymerases. The reaction mixture contained primer-template complexs (200 fmol), 1.25 units of TNAl, 50 mM Tris-HCl pH 8.5, 60 mM KCl, 30 mM (NH4) 2SO4, 1 mM MgCl2, and only 0.1 mM dITP. FIG. 14 shows primer extension assays using Pfu DNA polymerase. Templates containing no deaminated base (A), a single uracil (B) , or a single hypoxanthine (C) were used. The first and second lanes in each panel exhibit 23-mer primer and 44-mer template, respectively. FIG. 15 shows superimposed uracil-sensing domains and molecular docking of hypoxanthine. Cα-tracing of superposed uracil-sensing domains of 0.313 A RSMD from KODl DNA polymerase (red; PDB code, IWNS) and Pfu DNA polymerase
(green; PDB code, 2JGU) . Hypoxanthine is represented in stick surrounded by transparent surface.
FIG. 16 shows primer extension assays using various archaeal family B-type DNA polymerases . Templates containing no deaminated base (A) , a single xanthine (B) , two successive xanthines (C) and four successive xanthines (D) were used. The first and second lanes in each panel exhibit 23-mer primer and 44-mer template, respectively.
FIG. 17 shows optimal conditions of the nucleotide hydrolysis activities of dITPase.
FIG. 18 shows dynamic effect on hydrolysis of nucleotide triphosphate by dITPase and dUTPase.
[Best Mode] According to a first aspect, the present invention provides a dITPase protein and their functional equivalents . More specifically, the present invention provides dITPase protein and their functional equivalents having the activity of enhancing DNA polymerase processivity. Preferably, the dITPase protein according to the present invention is the protein of SEQ ID NO: 1, and specifically, "functional equivalent" includes a protein for enhancing DNA polymerase activity having amino acids sequence shown in SEQ ID NO: 1, or amino acid sequence variants having amino acid substitutions in some or all of the protein for enhancing DNA polymerase, or amino acid additions or deletions in some of the protein for enhancing DNA polymerase.
Also, the present invention includes the protein synthesized in vitro from public amino acids sequence.
Also, the present invention includes the purified protein comprising amino acids sequence with greater than 55% similarity to amino acids sequence shown in SEQ ID NO: 1. Preferably, the peptides have greater than 60% sequence similarity. More preferably, the peptides have greater than 70% sequence similarity. Most preferably, the peptides have greater than 80% sequence similarity. In the context of the present invention, amino acids sequence with more than 55% similarity means at least 55% identical or conservatively replaced amino acid residues in a like position when aligned optimally allowing for up to 4 gaps with the proviso that in respect of each gap a total not more than 10 amino acid residues is affected.
The amino acid substitutions are preferably conservative substitutions. Examples of the conservative substitutions of naturally occurring amino acids include aliphatic amino acids (GIy, Ala, and Pro) , hydrophobic amino acids (lie, Leu, and VaI) , aromatic amino acids (Phe, Tyr, and Trp) , acidic amino acids (Asp, and GIu) , basic amino acids (His, Lys, Arg, GIn, and Asn) , and sulfur- containing amino acids (Cys, and Met) . The deletions of amino acids are located in a region which is not involved directly in enhancing the activity of DNA polymerization.
According to a second aspect, the present invention provides a nucleotide sequence encoding the protein of SEQ ID NO: 1. Specifically, the nucleotide sequence is SEQ ID NO: 2.
As a result of the degeneracy of the genetic code, nucleotide sequences encoding the protein of SEQ ID NO: 1 can be prepared diversely. As used herein, the term "nucleic acid sequence" is intended to include natural mRNA identified selective expression, complementary DNA (cDNA) sequence and equivalent nucleic acid sequence thereof. As used herein, the term "equivalent nucleic acid sequence" is intended to include sequences provided herein, sequences with allelic variation and with variation between species, or degenerate codon sequence. As used herein, the term "degenerate codon sequence" refers to a nucleic acid sequence, which is different from said naturally occurring sequence, but encodes a polypeptide having the same sequence as that of the polypeptide of SEQ ID NO: 1 disclosed in the present invention. As used herein, the term "sequences with allelic variation" or "a sequence wit h variation between species" refers to a nucleic acid sequence, which is different from said naturally occurring nucleic acid sequence, but encodes a polypeptide having practically the same functional feature as that of said polypeptide disclosed herein.
According to a third aspect, the present invention provides a recombinant vector comprising the gene of SEQ ID NO: 2. As used herein, the term "vector" means a nucleic acid molecule that can carry another nucleic acid bound thereto. As used herein, the term "expression vector" is intended to include a plasmid, cosmid or phage, which can synthesize a protein encoded by a recombinant gene carried by said vector. A preferred vector is a vector that can self-replicate and express a nucleic acid bound thereto.
According to a fourth aspect, the present invention provides a host cell transformed with said recombinant vector. As used herein, the term "transformation" means that foreign DNA or RNA is absorbed into cells to change the genotype of the cells. Host cells suitable for transformation include prokaryotic, fungal, plant and animal cells, but are not limited thereto. Most preferably, E. coli cells are used. Methods for culturing E. coli are well known in the art.
According to a fifth aspect, the present invention provides a method for producing the protein of SEQ ID NO: 1 using said host cell. Optional recombinant proteins for expression include human proteins, and mean all proteins to use for treating diseases, or to be applicable industrially.
According to a sixth aspect, the present invention provides a specific antibody against the protein of SEQ ID NO: 1, wherein the specific antibody is prepared by injecting the protein of SEQ ID NO: 1.
According to a seventh aspect, the present invention provides a method for enhancing DNA polymerization with the DNA polymerase by using said protein.
As used herein, the term "DNA polymerase" refers to an enzyme that synthesizes DNA m the 5' -> 3' direction from deoxynucleotide triphosphate using a complementary template DNA strand and a primer by successively adding nucleotide to a free 3'-hydroxyl group. The template strand determines the sequence of the added nucleotide by Watson-Crick base pairing.
With mixture of the protein of SEQ ID NO: 1 and a DNA polymerase, the rate of DNA replication has increased. Accordingly, with mixture of the protein of SEQ ID NO: 1 and a DNA polymerase in Polymerase Chain Reaction (PCR) , a target DNA can be amplified more efficiently. The method of mixing of the protein of SEQ ID NO: 1 and a DNA polymerase can be simultaneously or differentially. That is one method is that mixture of the protein of SEQ ID NO: 1 and a DNA polymerase are added to PCR reaction solution, and then PCR reaction can be performed by adding target DNA fragment, and one another method is that target DNA are added to premix solution including a DNA polymerase, and then PCR reaction can be performed by adding the protein of SEQ ID NO: 1.
Hereinafter, the present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are for illustrative purposes only and are not to be construed to limit the scope of the present invention.
[Mode for Invention]
[Example 1] Cloning of TNAl HAM-I genes and expression of recombinant proteins Strains and culture conditi ons
Thermococcus sp. NAl was isolated from a deep-sea hydrothermal vent area in the East Manus Basin. YPS medium was used to culture Thermococcus sp. NAl for DNA manipulation. Culture and strain maintenance were performed according to standard procedures. To prepare a seed culture of Thermococcus sp. NAl, YPS medium in a 25-ml serum bottle was inoculated with a single colony from a phytagel plate and cultured at 900C for 20 h. Seed cultures were used to inoculate 700 ml of YPS medium in an anaerobic jar and cultured at 90°C for 2O h. E. coli strain DH5α was used for plasmid propagation and nucleotide sequencing. E. coli strain BL21-CodonPlus (DE3) -RIL cells (Stratagene, LaJoIIa, CA) and the plasmid pET-24a(+) (Novagen, Madison, WI) were used for gene expression. E. coli strains were cultivated in Luria-Bertani medium with 50 μg/ml kanamycin at 37°C.
DNA manipulation and sequencing DNA manipulations were performed using standard procedures, as described by Sambrook and Russell [Sambrook, J. & Russell, D. W., Molecular cloning: a laboratory manual, 3rd ed., Cold Spring Harbor, N. Y. (2001)]. Genomic DNA of Thermococcus sp. NAl was isolated using a standard procedure. Restriction enzymes and other modifying enzymes were purchased from Promega (Madison, WI) . Small-scale preparation of plasmid DNA from E. coli cells was performed
V) using a plasmid mini-prep kit (Qiagen, Hilden, Germany) . DNA sequencing was performed using an ABI3100 automated sequencer, using a BigDye terminator kit (PE Applied Biosystems, Foster City, CA) .
Cloning and expression of TNAl HAM-I encoding gene The full length of Thermococcus sp. NAl HAM-I gene flanked by Ndel and Xhol sites was amplified by PCR using two primers and genomic DNA as a template. The sense primer: 5'-CG ACC CGG CAT ATG AGG CTG GCG TTC ATC ACT TC-3'
(SEQ ID NO: 3) , the underlined sequence is Ndel site and the anti-sense primer: 5'-CT CCA CAT CTC GAG TTT AAG GTT TTC
CTT TAG CCA C-3' (SEQ ID NO: 4), the underlined sequence is
Xhol site. The amplified fragment was digested with Ndel and Xhol, and ligated with pET-24a(+) digested with Ndel/Xhol. The ligate was transformed into E. coli DH5α. For sequencing analysis and expression, the resulting plasmid was transformed into E. coli BL21-CodonPlus (DE3) - RIL and E. coli Rosetta (DE3) pLysS strain, respectively. Overexpression of the gene was induced by addition of isopropyl-β-D-thiogalactopyranoside (IPTG) at the mid- exponential growth phase, follow by 3-h incubation at 37 °C . The cells were harvested by centrifugation (6000 x g at 4 °C for 20 min) and resuspended in 50 mM Tris-HCl buffer (pH 8.0) containing 0.1 M KCl and 10% glycerol. The cells were disrupted by sonication and harvested by centrifugation (20,000 x g at 4 °C for 30 min). The resulting supernatant
] 6 was treated in a column of TALON™ metal affinity resin (BD Bioscience Clontech, Palo Alto, California) , and washed with 10 mM imidazole (Sigma, St. Louis, Mo.) in a 50 mM Tris-HCl buffer (pH 8.0) containing 0.1 M KCl and 10% glycerol, and the enzyme was eluted with 300 mM imidazole in buffer. The pooled fractions were dialyzed into 50 mM Tris-HCl (pH 8.0) buffer containing 10% glycerol using centricon YM-10 (Millipore, Bedford, MA) .
The concentrations of proteins were determined by the colorimetric assay of Bradford (1976) . The purification degrees of the proteins were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis according to a standard method (Laemmli, 1970) .
Results
Through the genomic sequence analysis of Thermococcus sp. NAl, an open reading frame (555 bp) encoding a 21 kDa protein consisting of 184 amino acids was found. In a pair- wise alignment with other HAM-I like genes, the deduced amino acid sequence of HAM-I showed 79.0% identity with HAM-I from Thermococcus sp. OGL-20P (accession no. AAP45001), 78.0% identity with HAM-I from Thermococcus kodakaraensis KODl (accession no. YP_184524), and 46.0% identity with mjO226 from Methanocaldococcus jannaschii DSM 2661 (FIG. 1) .
The TNAl HAM-I gene amplified by PCR was cloned into pET-24a(+) and transformed into E. coli BL21- CodonPlus (DE3)-RIL, and then expressed. In SDS-PAGE analysis, the 95% purified enzyme revealed a single protein band with a molecular mass of 21 kDa, which was deduced amino acid sequence (FIG. 2).
[Example 2] Applications of PCR with TNAl HAM-I protein
Effects in enhancing the efficiency of PCR were examined, which appeared to overcome electrolytic activity of PCR in the presence of inosine triphosphate (ITP), the
TNAl HAM-I protein and a high fidelity DNA polymerase. 50 ng of TNAl HAM-I protein was added to PCR mixture with various high fidelity DNA polymerases, respectively. PCR mixture was consisted of 2.5U of DNA polymerase, 150 ng of genomic DNA from Thermococcus sp. NAl as a template, 10 pmole of each primer, 200 μM dNTPs and PCR reaction buffer.
To amplify a 2-15kb fragment from the genomic DNA of
Thermococcus sp. NAl, primers were designed (Table 1).
< Table 1 > PCR Primer sequences used in this invention
Figure imgf000021_0001
First, it was found that PCR amplification of high fidelity DNA polymerases was inhibited by relatively low concentration of dITP (FIG. 3) . dITP was automatically generated by deamination of dATP on PCR amplification and considered to be mainly responsible for decreasing PCR efficiency. To solve these problems, effects of TNAl HAM-I in addition to PCR reaction with high fidelity DNA polymerase were examined.
As shown in FIG. 4, PCR reaction with high fidelity DNA polymerase in presence of TNAl HAM-I protein could be amplified a target DNA more effectively than in absence of TNAl HAM-I protein. It was expected that elimination of dITP by TNAl HAM-I protein in PCR reaction solution would be more effective in PCR amplification due to inhibition of stalling .
[Example 3] Effects in enhancing PCR amplification yield by dITPase
(1) Experimental methods
Construction of mutant DNA polymerase genes Site-specific mutagenesis of TNAl DNA polymerase gene was carried out using QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) . The primers for the mutation in this invention are summarized in Table 2.
< Table 2 > The position of amino acid substitution and the primer sequences for mutation
Name of Position of Mutation primer sequence (5' -3') mutants amino acid substitution
TNAl
T Y7F Tyr→Phe CTCGACGTCGATTTCATCACAGAGGACGGAAAGC
(SEQ ID NO: 12)
T V93Q Val→Gln CACCCGCAGGACCAACCCGCAATCCGCGACAAGATAAGG
(SEQ ID NO: 13)
T FlIbE Phe→Glu CGACATACCCGAAGCCAAGCGCTACCTC
(SEQ ID NO: 14)
Pfu
P Y7F Tyr→Phe TTAGATGTGGATTTCATAACTGAAGAAGGAAAAC (SEQ ID NO: 15)
P V93O Val→Gln CATCCCCAAGATCAACCCACTATTAGAGAAAAAGTT (SEQ ID NO: 16) P_F116E Phe→Glu TACGATATTCCAGAAGCAAAGAGATACCTCATCGAC
( SEQ ID NO : 17 )
Expression and purification of mutant DNA polymerases Plasmids were transformed Into Escherichia coli BL21 Rosetta (DE3)pLysS. Overexpression of genes was induced by addition of isopropyl-β-D-thiogalactopyranoside (IPTG) at the mid-exponential growth phase, followed by additional 3 h incubation at 37 0C. Cells were then, harvested by centrifugation (6000 x g at 4 °C for 20 min) , and resuspended in 50 mM Tris-HCl buffer (pH 8.0) containing 0.1 M KCl and 10% glycerol, followed by sonication. The cell lysate was centrifuged (20,000 x g at 4 0C for 30 min), and crude samples were prepared by heat treatment at 80 0C for 20 min. The resulting supernatant was applied to a column of TALON metal affinity resin (BD Biosciences, Clontech, Palo Alto, CA) and washed with 10 mM imidazole (Sigma, St. Louis, MO) in 50 mM Tris-HCl buffer (pH 8.0) containing 0.1 M KCl and 10% glycerol, and the protein was eluted with 300 mM imidazole in the same buffer. The pooled fractions were dialyzed in storage buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM DTT, 1 mM EDTA, and 10% glycerol.
The protein concentration was determined by Bradford assay (Bradford MM, 1976, Anal Biochem 72:248-254) , and protein purity was examined using sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) according to standard procedures (Laemmli UK, 1970, Nature 227:680-685). dITP incorporation assay
Incorporation assay was carried out as described by Chung JH et al. with slight modification (Chung J. H. et al., 2001, Nucleic Acids Res 29:3099-3107). In brief, a 5'- 32P-labeled 23-mer primer was annealed to the underlined portion of the 44-mer template (Table 3) and the primer- template complex (200 fmol) was incubated at 75 0C for 15 min with 1.25 units of TNAl DNA polymerase in 120 mM Tris- HCl (pH 8.8), 60 mM KCl, 30 mM (NH4J2SO4, 1 mM MgCl2, and 0.1 mM dITP. The reaction mixtures for Pfu (Promega) and KODl (Novagen) DNA polymerases were modified using each manufacturer's buffer. After incubation, each sample was analyzed by 15% polyacrylamide gel electrophoresis followed by autoradiography.
Primer extension assay
Primer extension assay was performed as described by Fogg et al. with slight modification (Fogg MJ, Pearl LH, Connolly BA, 2002, Nature Struct Biol 9:922-927). In brief, templates (Table 3) containing uracil, hypoxanthine or xanthine were used.
2? < Table 3 > Oligonucleotides used as substrates in this invention
Name Oligonucleotide sequence (5' -3' )
5' -32P-labeled 23-oligonucleotide primer
Labeled GGGGAGCCGCTAGAGTCGACCTC primer
44-oligonucleotide templates
C_tem GGAGACAAGCTTGATTGCCTCGAGGTCGACTCTAGCGGCTCCCC lU_tem GGAGACAAGCTTGAUTGCCTCGAGGTCGACTCTAGCGGCTCCCC 2U_tem GGAGACAAGCTTGUUTGCCTCGAGGTCGACTCTAGCGGCTCCCC lH_tem GGAGACAAGCTTGAHTGCCTCGAGGTCGACTCTAGCGGCTCCCC 2H_tem GGAGACAAGCTTGHHTGCCTCGAGGTCGACTCTAGCGGCTCCCC lX_tem GGAGACAAGCTTGAXTGCCTCGAGGTCGACTCTAGCGGCTCCCC 2X_tem GGAGACAAGCTTGXXTGCCTCGAGGTCGACTCTAGCGGCTCCCC
U, uracil; H, hypoxanthine; X, xanthine
The primer-template complex (400 fmol) was incubated at 75 0C for 15 min with 1.25 units of TNAl DNA polymerase in 120 mM Tris-HCl (pH 8.8), 60 inM KCl, 30 mM (NH4) 2SO4, 1 mM MgCl2, and 0.25 mM of each dNTP. The reaction mixtures for rTaq (Takara) , Pfu (Promega) , Deep-vent (New England Biolabs, Inc., Beverly, Mass), Vent (New England Biolabs, Inc., Beverly, Mass), and KODl (Novagen) DNA polymerases were slightly modified using each manufacturer's buffer. After incubation, each sample was analyzed by 15% polyacrylamide gel electrophoresis followed by autoradiography.
PCR
PCR amplification of 2, 5, 8, 10, 12 kb targets from λ DNA was carried out by TNAl and Pfu DNA polymerases using manufacturer's buffer. TNAl DNA polymerase buffer consisted of 120 mM Tris-HCl (pH 8.8), 30 mM (NH4J2SO4, 60 mM KCl, and 1 mM MgCl2 After single 1 min denaturation step at 95 °C, 30 cycles of denaturation (20 sec at 94 0C) , annealing and extension (2 kb, 2 min; 5 kb, 3.5 min; 8 kb, 5 min; 10 kb,
6 min; 12 kb, 7 min at 72 °C) were performed, followed by a final 7 min extension at 72 °C. PCR products were analyzed using 0.8% agarose gel electrophoresis. The primers as previously reported were used to amplify 2, 5, 8, 10 and 12 kb fragment from λ DNA (Kim YJ, et al., 2007, J Microbiol Biotechnol 17:1090-1097).
Stuructural analysis of uracil-sensing domain of Pfu and KODl DNA polymerase
For the structural analysis of uracil-sensing domain, PDB ID, 2JGU and IWNS were used. A hypoxantine was snugly docked into into the binding pocket in the uracil-sensing domain using the "solid docking" module in QUANTA (Molecular Simulation, Inc.). The program considers an electrostatic and geometric complementarity in docking a guest molecule into a host molecule.
(2) Results
dITP inhibits PCR amplification of family B-type DNA polymerases It was found that PCR amplification of family B-type DNA polymerases from hyperthermophilic archaea was significantly inhibited by dITP whereas Taq, a family A- type DNA polymerase, was not (FIG. 6) . Biochemical and structural investigation of Pfu DNA polymerase to unveil the molecular basis for the inhibitory effect of dITP demonstrated that dITP was incorporated into newly synthesized strands in the iirst round of the PCR, and subsequently uracil-sensing domain would sense the incorporated hypoxanthine in the template, eventually stalling replication (FIG. 13, FIG. 14 and FIG. 15) .
The mechanistic explanation of sensing hypoxanthine
Now that family B-type DNA polymerases of hyperthermophilic archaea have been reported to sense uracil, it was postulated that the presence of hypoxanthine in the template could be also sensed by family B-type DNA polymerases, making the polymerases stalled at encountering a hypoxanthine, eventually leading to decreased efficiency of polymerization. To address the issue, incorporation assay using dITP as a sole dNTP was tested to see whether dITP could affect DNA polymerase activity itself. Consequently, Pfu, KODl and TNAl polymerases could replicate the template, indicating that the polymerase activity of Pfu, KODl and TNAl polymerases was not inhibited by dITP (FIG. 13) . On the other hand, it is worthy to note that there was intriguing difference between Pfu and the other DNA polymerases in the incorporation assay. As shown in FIG. 13, Pfu DNA polymerase stopped right after adding a hypoxanthine to pair with cytosine. It is known that the paring with cytosine is the most stable even though hypoxanthine can pair with all four natural bases. In contrast, KODl and TNAl DNA polymerases could replicate the template up to the end (44-mer) using only dITP as a sole dNTP. It was thought that KODl and TNAl DNA polymerases were relatively insensitive to the wobble base pairing between hypoxanthine and other bases while Pfu DNA polymerase extremely prefers hypoxanthine and cytosine base-paring. Regardless of the disparity in the incorporation pattern, it was obvious that DNA polymerase activity itself was not affected by the presence of dITP.
Then, primer extension assay was performed to test whether the presence of hypoxanthine in the DNA template was a cause for the inhibitory effect of dITP. Obviously, Pfu DNA polymerase was stalled at a DNA template with hypoxanthine, similar to stalling at uracil, raising the possibility that Pfu DNA polymerase can sense hypoxanthine in the DNA template. To elucidate the mechanism how the DNA polymerase sense hypoxanthine in the DNA template, structural and biochemical analysis was performed as followed. Consequently, we found that uracil-sensing domain of the DNA polymerase was responsible for recognizing hypoxanthine in that mutations at critical residues (Y7, V93, and F116) in uracil-sensing domain could get over the stalling in the presence of hypoxanthine (FIG. 14), consistent with the recent report (Gill S. et al . , 2007, J MoI Biol) . Molecular docking model supports the finding that hypoxanthine was well fitted to uracil sensing domain (FIG. 15) . The docking with uracil may be the most favorable than other bases with less free energy, however, the hole could hold hypoxanthine as well .
In conclusion, dITP seemed to be incorporated in the first round of PCR, and then sensed by uracil-sensing domain, stalling DNA polymerases at the encountering hypoxanthine. Consistent with the observation with, family B-type DNA polymerases were also stalled in the presence of xanthine. However, the stalling position at xanthine was different (FIG. 16) .
The stalling of family B-type polymerases depends on intrinsic extension rate
Even though it was anticipated that family B-type DNA polymerases would share the sensing mechanism towέird hypoxanthine based on the common inhibitory effect of dITP and the sequence similarities among their uracil-sensing domains (FIG. 7), the primer extension analysis with five family B-type DNA polymerases revealed that they can be divided into two groups according to the stalling pattern: a single uracil in the template is enough to stall Pfu, Vent and Deep-vent DNA polymerases while KODl and TNAl DNA polymerases were stalled by two successive uracils (FIG. 8) . Similarly, two successive hypoxanthines were required to stall TNAl DNA polymerase (FIG. 9) . This observation clearly shows that some family B-type polymerases are not stalled on encountering a deamxnated base in a single stranded DNA, which is not compatible with the previous report on the stalling of fami Iy-B type polymerases (Greagg MA et al., 1999, Proc Natl Acad Sci USA 96:9045-9050., Fogg MJ et al., 2002, Nature Struct Biol 9:922-921., Shuttleworth G et al., 2004, J MoI Biol 337:6214-634). Despite the disparity in the stalling pattern, alteration of residues
(Y7, V93, and F116) in uraci L-sensing domain of TNAl DNA polymerase alleviated the stalling at both two successive hypoxanthines (uracils) in the primer extension assay (FIG. 9B and 9C) , facilitating PCR amplification of TNAl DNA polymerase in the presence of dITP (FIG. 9D) . Therefore, the failure of sensing the deaminated bases in the mutant proteins was not caused by changes in PCR activities, implicating that the uracil-sensing domain in TNAl DNA polymerase is a bona-fide machinery for sensing two successive deaminated bases.
Then, why are some fami Ly B-type polymerases stalled by two successive deaminated bases and are some enzymes stalled by a single deaminated base in spite of their common ability to recognize deaminated bases? To elucidate the molecular mechanism underpinning the difference in the stalling pattern, we thoroughly compared residues forming uracil-sensing domain between the two groups, and found that most residues in lid, base, and side of the uracil- sensing pocket were identical except some residues (FIG. 7). In addition, the structures of the uracil-sensing domains from KODl and Pfu DNA polymerases are virtually identical to each other. It is well known that sometimes a single amino acid difference can generate considerable functional differences in homologous proteins. To investigate the effect of the subtle difference in the amino acid composition of uracil-sensing domains, therefore, we selected Arg35, Phe87 and Ala95 as mutation targets and replaced each residue in TNAl DNA polymerase with the corresponding residues in Pfu DNA polymerase. The purified mutant proteins (R35E, F87L and A95T) still required two successive deaminated bases to be stalled, similar to the wild type TNAl DNA polymerase (data not shown) . In addition, chimeric proteins generated by whole domain swapping of uracil-sensing domains or exonuclease domains between Pfu and TNAl DNA polymerases, as denoted in FIG. 10, exhibited similar stalling patterns to the corresponding wild type proteins. For example, a fusion protein with the uracil-sensing domain from Pfu DNA polymerase and the exonuclease/polymerase domain from NAl followed the pattern of TNAl DNA polymerase and vice versa (FIG. 10) . Consequently, it is reasonable to conclude that the discrepancy in the stalling pattern was neither brought about by uracil-sensing domain nor exonuclease domain. According to the reports on the DNA polymerases (Kim YJ et al., 2007, J Microbiol Biotechnol 17:1090-1097., Takagi M et al., 1997, Appl Environ Microbiol 63:4504- 4510) , TNAl and KODl DNA polymerases are superior to other DNA polymerases in extension rates. TNAl and KODl DNA polymerase has three times and six times higher extension rate than Pfu and Deep-Vent DNA polymerases, respectively. Thus, to examine the relationship between the stalling pattern and the extension rate, we carried out primer extension assay at various temperatures with a rational that the extension rate would be affected by the reaction temperature. Interestingly, TNAl and KODl DNA polymerases were stalled at as low as 45 °C by a single deaminated base, but the stalling disappeared at higher temperatures (FIG. 11) . Recently, we got a mutant of TNAl DNA polymerase with three times higher extension rate than the wild type protein. Interestingly, two successive deaminated bases were not enough to stall the mutant protein. The result converges to support the close correlation between the stalling pattern and the extension rate. The reason why the binding of the first deaminated base to uracil-sensing domain fails to stall TNAl and KODl polymerases would be that the binding energy was not enough to stall the fast- moving enzyme. However, the interaction between the first deaminate base and the uracil-sensing domain could play as a brake to retard the enzyme, and subsequently, the DNA polymerase with a decreased mobility is then stalled by the second deaminated base. This stalling mechanism of DNA polymerases may explain why DNA polymerases with higher extension rate require more deaminated bases to be stalled. In summary, the binding of uracil (hypoxantine) to uracil- sensing domain, which commonly occurs in family B-type DNA polymerases, does not necessarily lead to the stalling of enzymes. The stalling is highly likely to be determined by both the extension rate of DNA polymerase and the recognition of deaminated bases . On the other hand, even though DNA polymerases with high extension rates require two successive deaminated bases to be stalled, it does not necessarily mean how it works in vivo. As reported, DNA polymerase works in concert with various auxiliary factors such as PCNA, replication factor, helicase and so on. It is possible that the factors or the condition in vivo could change the micro-environment of the enzyme, enable the DNA polymerases to sense even one base.
Spontaneous generation of dITP during PCR amplification and biotechnological application of dITPase
The stalling of family B-type DNA polymerases by hypoxanthine is an underlying mechanism to explain the inhibitory effect of dITP on PCR amplification. According to the previous reports (Hogrefe HH et al.r 2001, Proc Natl Acad Sci USA 99:596-601., Cho Y et al. , 2007, Mar Biotechnol (In press)), dUTP generated during PCR amplification decreased the PCR efficiency, which could be overcome by supplementing dUTPase. Concomitant with the study on dUTP, the implication in the sensing of hypoxanthine led us to examine the generation rate of dITP during PCR amplification. As a result, it was found that c. a. 0.9% of dITP was spontaneously generated, similar to that of dUTP.
< Table 4 > The spontaneous generation of dITP and the removal by dITPase activity
Product yield, %
Reaction dATP dADP dAMP dITP conditions (19.2-19.6) (13.7-14.1) (7.7-7.9) (18.5-18.9)
DATP
None 99.00 0.80 0.20 0.00
10 cycled 93.83 4.42 1.15 0.17
20 cycled 84.63 10.53 3.79 0.93 dATP (÷dlTPase)
10 cycled 94.09 5.33 1.26 0.02
20 cycled 86.53 10.95 2.42 0.00
- The retention times is given in parentheses for each nucleotide.
- The PCR conditions are as follows: 0.10 mM dATP cycled alone or with 5.00 ng μl-1 native dITPase. - Twenty five cycles: 95 0C for 1 min, 95 0C for 20 s, 72 0C for 8 min, 72 0C for 7 min.
Even though the amount of generated dITP was not enough to completely block PCR amplification of family B- type DNA polymerases, the incorporation of dITP in the first run was problematic in getting the PCR amplification maximized at efficiency, providing a chance for transition mutation. To handle the issue, we tried to supplement dITPase activity to remove generated dITP. A HAMl-like protein homologue (TNA1__HAM1) from Thermococcus onnurineus NAl was cloned and expressed in E. coli. TNA1_HAM1 showed similarities to HAMl-like protein from Thermococcus sp. OGL-20P (AAP45001, 79%), Hamlp homolog {Thermococcus kodakarensis KODl, YP__184524) , mjO226 from Methanocaldococcus jannaschii DSM2661 (NP_247195, 48%) (Chung JH et al., 2001, Nucleic Acids Res 29:3099-3107). The purified protein (FIG. 12) showed hydrolyzing activity toward dITP but did not hydrolyze dUTP. As shown in Table 4, the purified TNA1_HAM1 could eliminate the generated dITP during PCR amplification, and enhanced PCR amplification yield by TNAl and Pfu DNA polymerases (FIG. 12) . The characterization of purified HAMIp (Chung JH et al., 2001, Nucleic Acids Res 29:3099- 3107) and dUTPase (Hogrefe HH et al., 2001, Proc Natl Acad Sci USA 99:596-601., Cho Y et al., 2007, Mar Blotechnol (In press) ) showed that the two enzymes are very specific to hypoxanthine/xanthine and uracil dNTP, respectively. Now that family B-type DNA polymerases are stalled on encountering hypoxanthine (uracil) in the template, it was expected that elimination of both dUTP and dITP would be more effective in PCR amplification. As expected, the combination of TNA1_HAM1 and dUTPase (Cho Y et al., 2007, Mar Biotechnol (In press) ) was even more effective than the supplementation of a single protein (FIG. 12).
Unfortunately, due to the discontinued supply of dXTP, the hydrolyzing activity of TNA1_HAM1 toward dXTP could not be performed. Besides, the experiments to determine the generation rate of dXTP during PCR amplification and the inhibitory effect of dXTP could not be carried out either. However, the synthesis of a DNA oligomer containing xanthine base enabled us to show that DNA polymerases were stalled at encountering xanthine although the underlying mechanism behind the stalling seems different from that of hypoxanthine and uracil. The stalling position was not same as that of hypoxanthine or uracil and the mutations in uracil-sensing domain could not reverse the stalling. Furthermore, Taq DNA polymerase, a family A-type DNA polymerase was also stalled in the presence of xanthine base. Nonetheless, the stalling of DNA polymerases implicates that dXTP can be inhibitory to PCR amplification. Therefore, the PCR yield by adding TNA1_HAM1 may be cumulative effects of hydrolyzing dITP and dXTP, considering that dGTP deamination could take place.
In conclusion, the recognition (sensing) of deaminated bases is an intrinsic property of family B-type DNA polymerases of hyperthermophilic archaea living at temperatures above 80 0C, preventing transition mutation of deaminated bases. However, the stalling of DNA polymerase seems to rely on the molecular mobility during DNA replication. To apply dITPase is a strategic, effective way to escape the inhibitory effect of dITP or dXTP in the PCR.
[Example 4] Optimal conditions of the nucleotide hydrolysis activities of dITPase
(1) Experimental methods The nucleotide hydrolysis activity of dITPase was analyzed in the pH range 7.0-11.5 with dITP as substrate. dITPase was incubated in various buffer at 70° C. Tris buffer was used for pH 7.0-9.0 and Glycine buffer for pH 9.0-11.5. The dependence of the nucleotide hydrolysis activity of dITPase on temperature was also investigated. Assays of a possible temperature effect were performed at various temperatures (70° C-IOO" C) . All reaction mixtures were analyzed using a Partisi L-10 SAX column (4.6x250 mm, Waters Co. ) .
(2) Results
In order to investigate the optimal conditions of reaction, the effect of pH and temperature were examined. The effects of pH on the nucleotide hydrolysis activities of dITPase were analyzed under various pH conditions with the substrate dITP (FIG. 17) . The nucleotide hydrolysis activity of dITPase has alkaline pH. In particular, the reaction rates under neutraL conditions were <10% of maximum (FIG. 17). Dependence of the nucleotide hydrolysis activities of dITPase on temperature was also investigated. The optimal reaction temperature of dITPase was 70° C, but the activities was greatly reduced above 90° C (FIG. 17) .
[Example 5] Analysis of kinetic parameters for hydrolysis of dNTP by dNTPase
(1) Experimental methods To determine kinetic parameter for dITP and dUTP hydrolysis by dITPase and dUTPase, the dNTP hydrolyzing activities was measured using various dITP and dUTP concentration. The reaction for dITPase was performed in 50 μl of reaction mixture containing IxGlycine buffer (50 mM Glycine, 1 mM MgCl2, 80 mM NaCl, 0.001% BSA, pH 10.0), dITP
(2, 4, 6, 8, 10, 12, 14, 16 μM) , and 0.07 ng dITPase. The reaction for dUTPase was carried out in 50 μl of reaction mixture containing lxTris-HCl buffer (120 mM Tri-HCl, 1 mM
MgCl2, 60 mM KCl, 30 mM (NH4) 2SO4, 0.001% BSA, pH 7.0), dUTP (20, 40, 60, 80, 100, 120, 140 μM) , and 9 ng dUTPase. Rates of hydrolysis of nucleotide triphosphate were determined by various concentrations of the substrates. Amounts of products were measured using a Partisil-10 SAX column
(4.6x250 mm, Waters Co.).
(2) Results
Hydrolysis reactions by dITPase and dUTPase were examined with substrates (dITP and dUTP) . dITP purified from Thermococcus onnurioeus NAl (TNAl) efficiently hydrolyzed dITP rather than dITP purified from Methonococcus jannaschii (MJ) . However, dUTPase from TNAl hydrolyzed dUTP more slowly. The kinetic parameters for hydrolysis of nucleotide triphosphate by dITPase and dUTPase were measured (FIG. 18, Table 5) . The hydrolytic efficiency (Kcat/KM) of dITPase from TNAl was >10 times higher than that from MJ.
< Table 5 > Kinetic parameters for hydrolysis of dNTP by dNTPases
Figure imgf000039_0001
[Example 6] HPLC Analysis of PCR reaction products
(1) Experimental methods The reaction mixture (50 μl) contains 100 mM dATP and lxBuffer (120 mM Tris-HCl, 1 mM MgCl2, 60 mM KCl, 30 mM (NH4)2SO4, and 0.01% BSA), and was analyzed after 12 kb- targeted PCR reaction. Thermal condition is same as PCR condition. dITPase (5 ng) was used as a same time to check its effect on removing by-product (dITP) . The reaction products were chromatographed on Partisil-10 SAX column
(4.6x250 mm, Waters Co.), equLLibrated with 7 mM KH2PO4 (pH
4.0) (A) Products were eluted (1 ml/min) with 0.5M KH2PO4 and 0.5 M H2SO4 (pH 5.4) (B), using the gradient 0% B for 5 min, 0-100% B for 20 min, 100-0% B for 5 min. Absorbance at
254 nm was monitored with a UV detector, and peak areas were integrated. Nucleotide standards were purchased from
Sigma Co. and Roche Co. and chromatographed in a similar fashion.
(2) Results
The amount of dITP, which is produced during PCR reaction, has been carried out by HPLC. The peak expected to dITP was detected at 7.7-7.9 min and it was removed when dITPase was added to PCR mixture. It was observed that 0.93% of dATP was converted to dITP during PCR reaction (Table 4) .
[Example 7] PCR-Enhancing Activity of dITPase and dUTPase
(1) Experimental methods
To determine the effects of dITPase and dUTPase on PCR, the enzymes (2 and 5 ng each) was added to amplifications of 2, 5, 8, 10, and 12 kb lambda DNA targets conducted with cloned TNAl DNA polymerase (20 ng) , using extension time (2, 3.5, 5, 6, and 7 min) . Enhancing activity was identified by a visible increase in product yield. Two step reaction was used to amplify each DNA fragment under following conditions: an initial denaturation step of 1 min at 950C, 25 cycles of amplification (20 s at 95°C, 2, 3.5, 5, 6, and 7 min at 720C), and a final extension period of 5 min at 720C.
(2) Results Oxcidative deamination of DNA primarily convertes DNA base amino groups to keto groups. Thus, adenine is converted to hypoxanthine (HX) , guanine to xanthine (X) , and cytosine to uracil (U) . Also, a decrease in the activities of DNA polymerase, which is belonging to Family type B (TNAl, Pfu, and KOD) seemed to be slightly reduced by dITP and dUTP (Kim et al . , 2007, unpublished data). They are produced by deamination of DNA base amino group under high temperature condition followed by a decrease in product yield. Increase in product yield was identified in the PCR reaction containing dITPase and/or dUTPase. In case of using dITPase and dUTPase Ln same reaction mixture, PCR products were more produced compared to using dITPase or dUTPase separately. It means that dITP and dUTP are produced during PCR reaction and both enzymes, dITPase and dUTPase, removed each nucleotide derivatives by oxidative deamination. Also, similar result was observed in the PCR with Pfu. However, dITPase seemed to be more effective in PCR with Pfu. It seems that there is different mechanism between TNAl and Pfu in sensing each nucleotide (FIG. 12) .
[industrial Applicability] As described above, dITPase according to the present invention could be used for reactions of DNA amplification which was demanded for high sensitivity and rapidity, as supporting DNA polymerase activity.

Claims

[CLAIMS]
[Claim l]
A protein of SEQ ID NO: 1 or a functional equivalent thereof.
[Claim 2]
A nucleic acid sequence encoding the protein of Claim 1.
[Claim 3]
The nucleic acid according to Claim 2, which is SEQ ID NO: 2
[Claim 4]
The protein according to Claim 1, wherein the protein has the activity of enhancing processivity of DNA polymerization.
[Claim 5]
A method for enhancing I)NA polymerization with a DNA polymerase by using the protein according to Claim 1 or Claim 4.
[Claim 6] A recombinant vector comprising the gene of SEQ ID NO: 2. [Claim 7]
A host cell transformed with the recombinant vector of claim 6. [Claim 8]
A method for producing the protein of SEQ ID NO: ] by using the host cell of claim 7 „
4] [Claim 9]
A specific antibody against the protein of SEQ ID NO: 1, wherein the specific antibody is prepared by injecting the protein of SEQ ID NO: 1.
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