A Single Highly Mutable Catalytic Site Amino Acid Is Critical for DNA Polymerase Fidelity
2001; Elsevier BV; Volume: 276; Issue: 7 Linguagem: Inglês
10.1074/jbc.m008701200
ISSN1083-351X
AutoresPremal H. Patel, Hisaya Kawate, Elinor T. Adman, Matthew N. Ashbach, Lawrence A. Loeb,
Tópico(s)Cancer Genomics and Diagnostics
ResumoDNA polymerases contain active sites that are structurally superimposable and conserved in amino acid sequence. To probe the biochemical and structure-function relationship of DNA polymerases, a large library (200,000 members) of mutant Thermus aquaticus DNA polymerase I (Taq pol I) was created containing random substitutions within a portion of the dNTP binding site (Motif A; amino acids 605–617), and a fraction of all selected active Taq pol I (291 out of 8000) was tested for base pairing fidelity; seven unique mutants that efficiently misincorporate bases and/or extend mismatched bases were identified and sequenced. These mutants all contain substitutions of one specific amino acid, Ile-614, which forms part of the hydrophobic pocket that binds the base and ribose portions of the incoming nucleotide. Mutant Taq pol Is containing hydrophilic substitution I614K exhibit 10-fold lower base misincorporation fidelity, as well as a high propensity to extend mispairs. In addition, these low fidelity mutants containing hydrophilic substitution for Ile-614 can bypass damaged templates that include an abasic site and vinyl chloride adduct ethenoA. During polymerase chain reaction, Taq pol I mutant I614K exhibits an error rate that is >20-fold higher relative to the wild-type enzyme and efficiently catalyzes both transition and transversion errors. These studies have generated polymerase chain reaction-proficient mutant polymerases containing substitutions within the active site that confers low base pairing fidelity and a high error rate. Considering the structural and sequence conservation of Motif A, it is likely that a similar substitution will yield active low fidelity DNA polymerases that are mutagenic. DNA polymerases contain active sites that are structurally superimposable and conserved in amino acid sequence. To probe the biochemical and structure-function relationship of DNA polymerases, a large library (200,000 members) of mutant Thermus aquaticus DNA polymerase I (Taq pol I) was created containing random substitutions within a portion of the dNTP binding site (Motif A; amino acids 605–617), and a fraction of all selected active Taq pol I (291 out of 8000) was tested for base pairing fidelity; seven unique mutants that efficiently misincorporate bases and/or extend mismatched bases were identified and sequenced. These mutants all contain substitutions of one specific amino acid, Ile-614, which forms part of the hydrophobic pocket that binds the base and ribose portions of the incoming nucleotide. Mutant Taq pol Is containing hydrophilic substitution I614K exhibit 10-fold lower base misincorporation fidelity, as well as a high propensity to extend mispairs. In addition, these low fidelity mutants containing hydrophilic substitution for Ile-614 can bypass damaged templates that include an abasic site and vinyl chloride adduct ethenoA. During polymerase chain reaction, Taq pol I mutant I614K exhibits an error rate that is >20-fold higher relative to the wild-type enzyme and efficiently catalyzes both transition and transversion errors. These studies have generated polymerase chain reaction-proficient mutant polymerases containing substitutions within the active site that confers low base pairing fidelity and a high error rate. Considering the structural and sequence conservation of Motif A, it is likely that a similar substitution will yield active low fidelity DNA polymerases that are mutagenic. polymerase I Thermus aquaticus wild-type polymerase chain reaction kilobase(s) Prolonged survival of individual species depends on the accurate transmission of genetic material from one generation to the next (1Welch D.M. Meselson M. Science. 2000; 288: 1211-1215Crossref PubMed Scopus (456) Google Scholar). However, in times of stress, the propensity to mutate and to rapidly create variants that can escape selection pressures facilitates survival of a small fraction of the original population (2Radman M. Matic I. Taddei F. Ann. N. Y. Acad. Sci. 1999; 870: 146-155Crossref PubMed Scopus (85) Google Scholar). Thus, evolution may be characterized by periods of high fidelity DNA replication, as well as by the presence of transient mutators, which have a selective growth advantage during adverse conditions (3Mao E.F. Lane L. Lee J. Miller J.H. J. Bacteriol. 1997; 179: 417-422Crossref PubMed Scopus (256) Google Scholar). Identifying mechanisms of generating potential mutators is crucial toward understanding the dynamic processes that govern evolution, as well as toward devising effective chemotherapeutic strategies against pathogenic bacteria (4Oliver A. Canton R. Campo P. Baquero F. Blazquez J. Science. 2000; 288: 1251-1254Crossref PubMed Scopus (1139) Google Scholar, 5LeClerc J.E. Li B. Payne W.L. Cebula T.A. Science. 1996; 274: 1208-1211Crossref PubMed Scopus (665) Google Scholar) and cells (6Loeb L.A. Lindahl T. Genetic Instability in Cancer. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 329-342Google Scholar) that mutate at elevated rates.Cells have evolved multistep mechanisms to guarantee the exceptionally high fidelity of DNA replication that is required for the maintenance of species. The genetic sequence of organisms is maintained over prolonged evolution by the fidelity of DNA replication (7Kornberg A. Baker T. DNA Replication. W. H. Freeman and Co., New York1992Google Scholar), the efficiency of DNA repair processes (8Lindahl T. Nyberg B. Biochemistry. 1972; 11: 3610-3618Crossref PubMed Scopus (1161) Google Scholar), and the recombination-mediated lateral transfer events (9Patel P.H. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5095-5100Crossref PubMed Scopus (85) Google Scholar). Quantitatively, nucleotide selection at the active site of DNA polymerases is the most significant contributor to the fidelity of DNA replication (10Kunkel T.A. Loeb L.A. Science. 1981; 213: 765-767Crossref PubMed Scopus (127) Google Scholar). Nucleotide selection includes correct Watson-Crick base pair formation between complementary bases; further discrimination of base selection occurs by a conformational change at the active site during each nucleotide addition step (11Johnson K.A. Annu. Rev. Biochem. 1993; 62: 685-713Crossref PubMed Scopus (504) Google Scholar) and preferential extension of the correct base pair by the addition of the next complementary nucleotide (12Perrino F.W. Preston B.D. Sandell L.L. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8343-8347Crossref PubMed Scopus (120) Google Scholar). Together, these processes contribute ∼100,000-fold to the overall accuracy of DNA replication (one base change per 108–10 bases per generation (13Drake J.W. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7160-7164Crossref PubMed Scopus (843) Google Scholar)). Inefficient extension of mispaired bases in vivo would facilitate 3′-5′ exonuclease removal of the nascent nucleotide. Exonucleolytic (3′-5′) proofreading activity of most DNA polymerases occurs on a separate domain (alternatively, this activity could reside in a separate protein) and contributes, on average, 10-fold to the overall mutation rate (14Echols H. Lu C. Burgers P.M. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2189-2192Crossref PubMed Scopus (110) Google Scholar). In addition, errors in catalysis by DNA polymerases are subsequently corrected by a mismatch repair system, which contributes an additional 2–3 orders of magnitude to the overall accuracy of DNA replication (15Modrich P. J. Biol. Chem. 1989; 264: 6597-6600Abstract Full Text PDF PubMed Google Scholar). Disruption of either mismatch repair system or polymerase 3′-5′ exonuclease function within cells leads to a mutator phenotype (16Fishel R. Lescoe M.K. Rao M.R.S. Copeland N.G. Jenkins N.A. Garber J. Kane M. Kolodner R. Cell. 1993; 75: 1027-1038Abstract Full Text PDF PubMed Scopus (2584) Google Scholar, 17Bronner C.E. Baker S.M. Morrison P.T. Warren G. Smith L.G. Lescoe M.K. Kane M. Earabino C. Lipford J. Lindblom A. Tannergard P., R.J., B. Godwin A.R. Ward D.C. Nordenskjold M. Fishel R. Kolodner R. Liskay R.M. Nature. 1994; 368: 258-261Crossref PubMed Scopus (1916) Google Scholar). Mice harboring disruption in mismatch repair (18Reitmair A.H. Schmits R. Ewel A. Bapat B. Redston M. Mitri A. Waterhouse P. Mittrucker H.W. Wakeham A. Liu B. Thomason A. Griesser H. Gallinger S. Ballhausen W.G. Fishel R. Mak T.W. Nat. Genet. 1995; 11: 64-70Crossref PubMed Scopus (348) Google Scholar) or in the 3′-5′ exonuclease of DNA polymerase δ develop cancer in multiple organs with elevated frequency. 1B. Preston, personal communication.1B. Preston, personal communication. These studies provide direct evidence linking deficits in the fidelity of DNA synthesis with increased incidence of cancer.The structure of a DNA polymerase resembles the human right hand and contains three distinct subdomains (finger, palm, and thumb (19Beese L.S. Derbyshire V. Steitz T.A. Science. 1993; 260: 352-355Crossref PubMed Scopus (450) Google Scholar)). High resolution crystal structures of DNA polymerase within the pol I2 family of enzymes indicate that the base of the incoming nucleotide stacks with the hydrophobic planar amino acids located in the fingers subdomain (Motif B), and the triphosphate portion is bonded through metal cations by ionic interactions with Asp-610 located in the palm subdomain (Motif A (20Doublie S. Tabor S. Long A.M. Richardson C.C. Ellenberger T. Nature. 1998; 391: 251-258Crossref PubMed Scopus (1097) Google Scholar,21Li Y. Korolev S. Waksman G. EMBO J. 1998; 17: 7514-7525Crossref PubMed Scopus (653) Google Scholar)). During nucleotide incorporation, DNA polymerases undergo a conformation change from open to a closed conformation, bringing the fingers subdomain in close proximity to the palm subdomain (21Li Y. Korolev S. Waksman G. EMBO J. 1998; 17: 7514-7525Crossref PubMed Scopus (653) Google Scholar). Planar hydrophobic residues of the fingers subdomain sense the binding of a properly templated incoming nucleotide, and this signal is transduced to the catalytic residues of the palm subdomain (22Patel P.H. Preston B.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 549-553Crossref PubMed Scopus (142) Google Scholar). Among those residues that are located between the base and phosphate interacting amino acids and can participate in transducing this signal are the highly conserved DYSQIELR Motif A residues (in Taq pol I, amino acids 605–617). The nucleotides encoding these amino acids are conserved within DNA polymerase I of all prokaryotes and eubacteria sequenced (Fig. 1) (9Patel P.H. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5095-5100Crossref PubMed Scopus (85) Google Scholar). The amino acids in this region are positioned to have a potentially important contribution toward DNA polymerase fidelity.The high contribution of the polymerase active site to the overall fidelity of DNA synthesis suggests that subtle alterations within the catalytic site should lead to polymerases with lower fidelity. This is especially true for polymerases lacking a 3′-5′ proofreading exonuclease activity that can excise misincorporated nucleotides. Site-directed mutagenesis studies have identified a conserved tyrosine located within the dNTP-binding pocket that, when substituted to nonplanar amino acids, reduce the fidelity of the polymerase by 5–10-fold, but these mutant polymerases also exhibit markedly reduced catalytic activity (23Carroll S.S. Cowart M. Benkovic S.J. Biochemistry. 1991; 30: 804-813Crossref PubMed Scopus (116) Google Scholar, 24Joyce C.M. Sun X.C. Grindley N.D. J. Biol. Chem. 1992; 267: 24485-24500Abstract Full Text PDF PubMed Google Scholar, 25Desai, S. D., Pandey, V. N., and Modak, M. J. (1994)Biochemistry 11868–11874Google Scholar). Previously, we established a library of ∼8000 different active Taq DNA polymerase mutants using random sequence mutagenesis and stringent selection protocol. Each mutant contained one or more substitutions within Motif A and maintained 10–200% of the wild-type activity. In this study, we screened 291 different mutant DNA polymerases containing substitutions within the dNTP binding site for altered polymerase fidelity. Many of the low fidelity mutants contained multiple substitutions; however, each contained a substitution at a single position. Mutants containing substitutions of one residue (Ile within the conserved DYSQIELR sequence), which can be substituted to diverse amino acids, yield an enzyme that introduces transition and transversion errors 20-fold more efficiently than the WT Taq polymerase. Alterations at the other sites did not have a significant effect on fidelity. Because this residue is conserved in structure and sequence in polymerases from prokaryotes, eukaryotes, and archea (26Delarue M. Poch O. Tordo N. Moras D. Argos P. Protein Eng. 1990; 3: 461-467Crossref PubMed Scopus (573) Google Scholar, 27Wang J. Sattar A.K. Wang C.C. Karam J.D. Konigsberg W.H. Steitz T.A. Cell. 1997; 89: 1087-1099Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar), it represents a potentially important target for the creation of mutator DNA polymerases.DISCUSSIONThe accuracy of DNA replication is crucial for maintaining genomic stability from one generation to the next (37Echols H. Biochimie (Paris). 1982; 64: 571-575Crossref PubMed Scopus (30) Google Scholar, 38Loeb L.A. Kunkel T.A. Annu. Rev. Biochem. 1982; 51: 429-457Crossref PubMed Scopus (367) Google Scholar). Nevertheless, in times of crisis, it is beneficial for cells to exhibit diversity and thus mutate at higher rates. The fidelity of DNA replication is largely determined at the DNA polymerase active site, which is responsible for 5–6 orders of magnitude of the overall mutation rate of cells (11Johnson K.A. Annu. Rev. Biochem. 1993; 62: 685-713Crossref PubMed Scopus (504) Google Scholar). Thus far, the majority of the bacteria populations that mutate at high rates, which have been investigated, contain loss of specific DNA repair pathways (39Miller J.H. Mutat. Res. 1998; 409: 99-106Crossref PubMed Scopus (55) Google Scholar). Studies with mutant DNA polymerases have mainly focused on analyzing the effects after the loss of 3′-5′ exonucleolytic proofreading activity. E. coli and yeast harboring DNA polymerases with loss of exonucleolytic activity exhibit a 10-fold elevated mutation rate (14Echols H. Lu C. Burgers P.M. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2189-2192Crossref PubMed Scopus (110) Google Scholar, 40Schaaper R.M. Genetics. 1989; 121: 205-212Crossref PubMed Google Scholar). Development of additional mutants, particularly those containing substitutions within the active site of polymerases, coupled with high resolution crystal structures, should advance our understanding of the determinants of polymerase accuracy, as well as facilitating studies of the phenotypes associated with mutated polymerases exhibiting poor fidelity. However, current structure-based site-directed mutagenesis studies have not been successful at producing mutant polymerases with WT-like activity that exhibit low fidelity.We have found, following random mutagenesis of a portion of the polymerase active site and stringent selection, a single amino acid residue (Ile-614) that, when substituted to a variety of hydrophilic amino acids, reduces the fidelity by at least 10-fold. No other amino acid within Motif A, when substituted, consistently exhibited such low base pairing fidelity. In addition, nonhydrophilic substitutions, including mutant I614M, did not alter the error rate during DNA synthesis (Table V). In reactions containing 3 nucleotides (Fig. 2), mutants containing substitutions at Ile-614 are able to misincorporate a base opposite the template position for which there is not a complementary dNTP. In addition, these mutant polymerases can also extend nascent primers containing mismatched DNA termini more efficiently than can WT Taq pol I. The ability for polymerases containing hydrophilic substitutions at position 614 to efficiently catalyze misincorporation was tested kinetically. Mutant I614K was shown to misincorporate nucleotides 10-fold more efficiently relative to WT enzyme; in addition, kinetic experiments showed that I614K mutant is also efficient at forming transversion errors by misextending pyrimidine-pyrimidine base pairs at higher rates relative to WT. These kinetic data suggest the mutant I614K Taq pol I should produce both transition and transversion errors, and WTTaq pol I and mutant 53 (I614K) should exhibit unique error spectrums following DNA replication of a specific sequence. We tested these predictions by conducting PCR amplification of a homogeneous sequence and measured the spectrum of mutations produced by mutant 53 (I614K). The results show that whereas WT Taq pol I and mutant 346 (A608D/E615D) contain very similar distribution of errors and that these errors mirror the published spectrum of WTTaq pol I (36Tindall K.R. Kunkel T.A. Biochemistry. 1988; 27: 6008-6013Crossref PubMed Scopus (624) Google Scholar), mutant 53 (I614K) exhibits markedly elevated transversion errors, especially A:T → T:A and an error rate that is 20 times higher than WT Taq pol I. Interestingly, mutation spectrum of WT Taq pol I under mutagenic conditions in the presence of Mn2+ resembles that of mutant 53 (I614K) under normal conditions, although the error rate of mutant 53 is higher than that of Mn2+ catalyzed WT enzyme. 3P. H. Patel, H. Kawate, E. Adman, M. Ashbach, and L. A. Loeb, unpublished results.The fidelity of mutant Taq polymerase containing a hydrophilic substitution at Ile-614 is comparable to UmuC class of polymerase (34Johnson R.E. Washington M.T. Prakash S. Prakash L. J. Biol. Chem. 2000; 275: 7447-7450Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar, 35Tang M. Shen X. Frank E.G. O'Donnell M. Woodgate R. Goodman M.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8919-8924Crossref PubMed Scopus (483) Google Scholar). This class of polymerases contains members with modest DNA polymerase activity, yet these polymerases are particularly adept at bypassing template lesions (for reviews, see Refs. 41Friedberg E.C. Gerlach V.L. Cell. 1999; 98: 413-416Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar and 42Goodman M.F. Trends Biochem Sci. 2000; 25: 189-195Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). We find hydrophilic substitutions at Ile-614 result in highly active DNA polymerases that can also bypass damaged templates (abasic site and replication blocking vinyl chloride adduct ethenoA). In addition, hydrophilic substitutions at Ile-614 facilitate the incorporation of bulky fluorescent nucleotide analogs,3 and a wide variety of hydrophobic and hydrophilic substitutions at Ile-614 allow successive rNTP incorporation and synthesis of RNA (49Patel P.H. Loeb L.A. J. Biol. Chem. 2000; 275: 40266-40272Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The low fidelity, the ability to bypass template lesions, and an error spectrum that parallels incubations in the presence of manganese suggest that hydrophilic substitutions for Ile-614 should lead to a "wider" active site.Amino acid Ile-614 is located in a highly conserved DYSQIELR Motif A sequence; this Ile residue is maintained in large majority of prokaryotic pol I class of enzymes (Fig. 1). Interestingly, the Motif A nucleotide sequence is highly conserved within individual prokaryotic species of diverse genera (e.g. Thermus, Rickettsia, and Mycobacteria). High resolution x-ray crystal structure of Taq pol I complexed with DNA and an incoming nucleotide triphosphate suggests that three hydrophobic amino acids (Ile-614, Phe-667, and Tyr-671) pack near the ribose and base portions of the incoming nucleotide (Fig.5). Substitution of the homologous Tyr to a nonplanar amino acid within E. coli pol I (23Carroll S.S. Cowart M. Benkovic S.J. Biochemistry. 1991; 30: 804-813Crossref PubMed Scopus (116) Google Scholar, 24Joyce C.M. Sun X.C. Grindley N.D. J. Biol. Chem. 1992; 267: 24485-24500Abstract Full Text PDF PubMed Google Scholar) and mammalian DNA pol α (43Dong Q. Copeland W.C. Wang T.S. J. Biol. Chem. 1993; 268: 24163-24174Abstract Full Text PDF PubMed Google Scholar) yields enzymes with 10-fold lower fidelity (and reduced catalytic activity). In the closed Taq pol I-DNA-ddNTP ternary form of the structure, this residue is not in contact with the nucleotide, but instead hydrogen bonds to Glu-615. In the open ternary form, this Tyr residue occupies the site of the template base, opposing the incoming nucleotide and hydrogen bonded to it. Substitutions of Phe-667 to Tyr within Taq pol I yields active enzymes capable of incorporating dideoxynucleotides (44Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6339-6343Crossref PubMed Scopus (300) Google Scholar). In the closed form of Taq pol I, Phe-667 is >3.7 Å from the base, but in the open form, this residue packs near the ribose, the base, and the middle phosphate oxygen of the incoming nucleotide. Ile-614 packs against the ribose ring and the other free oxygen of the middle phosphate in both the closed and open forms of the Taq pol I-DNA-ddNTP ternary structure (21Li Y. Korolev S. Waksman G. EMBO J. 1998; 17: 7514-7525Crossref PubMed Scopus (653) Google Scholar). We propose that diverse substitutions for Ile-614, and especially hydrophilic substitutions, lead to a more "open" pocket that can accommodate damaged templates, non-Watson-Crick base pairs, and diverse nucleotide analogs. This model proposes that stable stacking/packing interactions with the base and ribose rings are crucial for polymerase fidelity and is consistent with a model for nucleotide incorporation proposed for HIV-1 reverse transcriptase (45Patel P.H. Jacobo-Molina A. Ding J. Tantillo C. Clark Jr., A.D. Raag R. Nanni R.G. Hughes S.H. Arnold E. Biochemistry. 1995; 34: 5351-5363Crossref PubMed Scopus (176) Google Scholar) and other polymerases (46Goodman M.F. Fygenson K.D. Genetics. 1998; 148: 1475-1482Crossref PubMed Google Scholar).Figure 5Residues from Motif A in contact with incoming nucleotide. At different stages of the binding of the incoming nucleotide, Ile-614, Phe-667, and Tyr-671, and the aliphatic portion of Glu-615 contact the incoming nucleotide. Shown is the closed form of the Taq pol I-DNA-ddCTP complex, emphasizing the packing of Ile-614 (yellow). The metal ions are dark gray. Hydrophilic substitutions for Ile-614 residue result in polymerases with high DNA pol activity and can misincorporate nucleotides with a very high efficiency. Additional interactions (not shown) include triphosphate interactions with O-helix residues Lys and Arg and the interactions with Phe-667 and Tyr-671, which differ between open and closed forms of the complex. This drawing was made of salient residues from Taq pol I structure determined by Li et al. (PDB code 3ktq) (21Li Y. Korolev S. Waksman G. EMBO J. 1998; 17: 7514-7525Crossref PubMed Scopus (653) Google Scholar) by E. Adman using MOLSCRIPT (47Kraulis P. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar) and Raster3D (48Merritt E.A. Murphy M.E.P. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 869-873Crossref PubMed Scopus (2854) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)In summary, we have evolved a set of polymerases containing substitutions at a single amino acid with low base pairing fidelity, the ability to bypass template lesions, and the ability to incorporate nucleotide analogs. Substitution of Ile-614, an amino acid that is structurally conserved in all DNA polymerases, produces active DNA polymerase with very broad substrate specificity. These findings are consistent with models of adaptive evolution that 1) in times of stress, the inherent plasticity of enzyme active site facilitates the generation of beneficial mutants with altered substrate specificity, which could provide a selective advantage, and 2) following successful survival through periods of adverse conditions, WT nucleotide sequence (one that is fit and the most prevalent) can be generated through recombination-mediated lateral transfer (9Patel P.H. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5095-5100Crossref PubMed Scopus (85) Google Scholar). This study suggests that other populations of mutators may contain substitutions within the polymerase catalytic site that confer low fidelity. Considering the vital role of DNA polymerases during DNA replication, repair, and recombination, it may be important to genotype tumors characterized by elevated mutation rates for polymorphic differences within the polymerase catalytic site. Prolonged survival of individual species depends on the accurate transmission of genetic material from one generation to the next (1Welch D.M. Meselson M. Science. 2000; 288: 1211-1215Crossref PubMed Scopus (456) Google Scholar). However, in times of stress, the propensity to mutate and to rapidly create variants that can escape selection pressures facilitates survival of a small fraction of the original population (2Radman M. Matic I. Taddei F. Ann. N. Y. Acad. Sci. 1999; 870: 146-155Crossref PubMed Scopus (85) Google Scholar). Thus, evolution may be characterized by periods of high fidelity DNA replication, as well as by the presence of transient mutators, which have a selective growth advantage during adverse conditions (3Mao E.F. Lane L. Lee J. Miller J.H. J. Bacteriol. 1997; 179: 417-422Crossref PubMed Scopus (256) Google Scholar). Identifying mechanisms of generating potential mutators is crucial toward understanding the dynamic processes that govern evolution, as well as toward devising effective chemotherapeutic strategies against pathogenic bacteria (4Oliver A. Canton R. Campo P. Baquero F. Blazquez J. Science. 2000; 288: 1251-1254Crossref PubMed Scopus (1139) Google Scholar, 5LeClerc J.E. Li B. Payne W.L. Cebula T.A. Science. 1996; 274: 1208-1211Crossref PubMed Scopus (665) Google Scholar) and cells (6Loeb L.A. Lindahl T. Genetic Instability in Cancer. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 329-342Google Scholar) that mutate at elevated rates. Cells have evolved multistep mechanisms to guarantee the exceptionally high fidelity of DNA replication that is required for the maintenance of species. The genetic sequence of organisms is maintained over prolonged evolution by the fidelity of DNA replication (7Kornberg A. Baker T. DNA Replication. W. H. Freeman and Co., New York1992Google Scholar), the efficiency of DNA repair processes (8Lindahl T. Nyberg B. Biochemistry. 1972; 11: 3610-3618Crossref PubMed Scopus (1161) Google Scholar), and the recombination-mediated lateral transfer events (9Patel P.H. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5095-5100Crossref PubMed Scopus (85) Google Scholar). Quantitatively, nucleotide selection at the active site of DNA polymerases is the most significant contributor to the fidelity of DNA replication (10Kunkel T.A. Loeb L.A. Science. 1981; 213: 765-767Crossref PubMed Scopus (127) Google Scholar). Nucleotide selection includes correct Watson-Crick base pair formation between complementary bases; further discrimination of base selection occurs by a conformational change at the active site during each nucleotide addition step (11Johnson K.A. Annu. Rev. Biochem. 1993; 62: 685-713Crossref PubMed Scopus (504) Google Scholar) and preferential extension of the correct base pair by the addition of the next complementary nucleotide (12Perrino F.W. Preston B.D. Sandell L.L. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8343-8347Crossref PubMed Scopus (120) Google Scholar). Together, these processes contribute ∼100,000-fold to the overall accuracy of DNA replication (one base change per 108–10 bases per generation (13Drake J.W. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7160-7164Crossref PubMed Scopus (843) Google Scholar)). Inefficient extension of mispaired bases in vivo would facilitate 3′-5′ exonuclease removal of the nascent nucleotide. Exonucleolytic (3′-5′) proofreading activity of most DNA polymerases occurs on a separate domain (alternatively, this activity could reside in a separate protein) and contributes, on average, 10-fold to the overall mutation rate (14Echols H. Lu C. Burgers P.M. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2189-2192Crossref PubMed Scopus (110) Google Scholar). In addition, errors in catalysis by DNA polymerases are subsequently corrected by a mismatch repair system, which contributes an additional 2–3 orders of magnitude to the overall accuracy of DNA replication (15Modrich P. J. Biol. Chem. 1989; 264: 6597-6600Abstract Full Text PDF PubMed Google Scholar). Disruption of either mismatch repair system or polymerase 3′-5′ exonuclease function within cells leads to a mutator phenotype (16Fishel R. Lescoe M.K. Rao M.R.S. Copeland N.G. Jenkins N.A. Garber J. Kane M. Kolodner R. Cell. 1993; 75: 1027-1038Abstract Full Text PDF PubMed Scopus (2584) Google Scholar, 17Bronner C.E. Baker S.M. Morrison P.T. Warren G. Smith L.G. Lescoe M.K. Kane M. Earabino C. Lipford J. Lindblom A. Tannergard P., R.J., B. Godwin A.R. Ward D.C. Nordenskjold M. Fishel R. Kolodner R. Liskay R.M. Nature. 1994; 368: 258-261Crossref PubMed Scopus (1916) Google Scholar). Mice harboring disruption in mismatch repair (18Reitmair A.H. Schmits R. Ewel A. Bapat B. Redston M. Mitri A. Waterhouse P. Mittrucker H.W. Wakeham A. Liu B. Thomason A. Griesser H. Gallinger S. Ballhausen W.G. Fishel R. Mak T.W. Nat. Genet. 1995; 11: 64-70Crossref PubMed Scopus (348) Google Scholar) or in the 3′-5′ exonuclease of DNA polymerase δ develop cancer in multiple organs with elevated frequency. 1B. Preston, person
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