DNA Replication Fidelity

When describing the structure of the DNA double helix, Watson and Crick (1Watson J.D. Crick F.H.C. Nature. 1953; 171: 737-738Crossref PubMed Scopus (8554) Google Scholar) wrote, "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." Fifty years later, interest in the fidelity of DNA copying mechanisms remains high because the balance between correct and incorrect DNA synthesis is relevant to a great deal of biology. High fidelity DNA synthesis is beneficial for maintaining genetic information over many generations and for avoiding mutations that can initiate and promote human diseases such as cancer and neurodegenerative diseases. Low fidelity DNA synthesis is beneficial for the evolution of species, for generating diversity leading to increased survival of viruses and microbes when subjected to changing environments, and for the development of a normal immune system. What was not yet appreciated 50 years ago was the large number and amazing diversity of transactions involving DNA synthesis required to faithfully replicate genomes and to stably maintain them in the face of constant challenges from cellular metabolism and the external environment. To perform these tasks, cells harbor multiple DNA polymerases (2Hübscher U. Maga G. Spadari S. Annu. Rev. Biochem. 2002; 71: 133-163Crossref PubMed Scopus (588) Google Scholar, 3Shcherbakova P.V. Bebenek K. Kunkel T.A. Sci. Aging Knowl. Environ..sageke.sciencemag.org/cgi/content/full/sageke;2003/8/ref31Date: 2003Google Scholar), many of which have only been discovered in the past 5 years and whose cellular functions are not fully understood. These polymerases differ in many features including their fidelity. This diversity and the sequence complexity of genomes provide the potential to vary DNA synthesis error rates over a wider range than was appreciated a few years ago. This article reviews major concepts and recent progress on DNA replication fidelity with additional perspectives found in longer reviews cited throughout. Studies of bacteriophage and Escherichia coli replication in the absence of DNA mismatch repair and external environmental stress suggest that the base substitution error rate of the replication machinery in vivo is in the range of 10–7 to 10–8 (4Schaaper R.M. J. Biol. Chem. 1993; 268: 23762-23765Abstract Full Text PDF PubMed Google Scholar). Eukaryotic DNA replication is likely to be at least this accurate (5Loeb L.A. Cancer Res. 2003; 51: 3075-3079Google Scholar). High chromosomal replication fidelity in vivo is matched in vitro by the accuracy of E. coli and human replication complexes and replicative polymerases that have intrinsic proofreading exonuclease activities (Fig. 1, top left). Error rates during DNA synthesis are in the 10–6 to 10–8 range for replicative polymerases in family A (e.g. T7 Pol), family B (e.g. T4 Pol, Pol δ, Pol ϵ) and family C (e.g. E. coli Pol III). Comparisons with error rates for their proofreading-defective derivatives (Fig. 1, designated A–, B–, C–, and RT–, for viral reverse transcriptase) reveal that high fidelity typically results from 104 to 106-fold polymerase selectivity for inserting correct rather than incorrect nucleotides, followed by excision of 90–99.9% of base-base mismatches by exonucleases that are either intrinsic to the polymerase (e.g. T7 Pol, T4 Pol, Pol δ, Pol ϵ) or encoded by a separate gene (e.g. the ϵ subunit of E. coli Pol III). Genome stability also requires the ability to repair DNA damage that comes in many forms and is repaired by several different pathways (6Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology, Washington, D. C.1995Google Scholar), most of which require DNA synthesis to fill gaps created when lesions are excised. Error rates for repair reactions have not yet been extensively studied. Gap filling during mismatch repair, nucleotide excision repair, and long patch base excision repair (BER) 1The abbreviations used are: BER, base excision repair; TLS, translesion synthesis. is performed by A and B family polymerases with intrinsic proofreading activity. Thus, these repair reactions are predicted to be accurate, consistent with known roles in suppressing damage-induced mutagenesis. Repair requiring filling gaps of one or a few nucleotides, such as "short patch" BER and repair of DNA double strand breaks by non-homologous end joining, use family X polymerases. On average, these are less accurate than replicative polymerases (Fig. 1) partly, but not exclusively, due to a lack of intrinsic proofreading. Lesions that escape repair can potentially reduce replication fidelity. Translesion synthesis (TLS) polymerases copy past lesions in DNA that block the major replicative polymerases (7Goodman M.F. Annu. Rev. Biochem. 2002; 71: 17-50Crossref PubMed Scopus (628) Google Scholar, 8Kunkel T.A. Pavlov Y.I. Benenek K. DNA Repair. 2003; 2: 135-149Crossref PubMed Scopus (118) Google Scholar, 9Friedberg E.C. Wagner R. Radman M. Science. 2002; 296: 1627-1630Crossref PubMed Scopus (393) Google Scholar, 10Prakash S. Prakash L. Genes Dev. 2002; 16: 1872-1883Crossref PubMed Scopus (295) Google Scholar, 11Livneh Z. J. Biol. Chem. 2001; 276: 25639-25642Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). One is the B family member Pol ζ, and others are in the Y family, members of which are found in organisms from bacteria to man (e.g. E. coli Pol IV and V and mammalian Pol η, Pol ι, and Pol κ). Also lacking proofreading activity, these are the least accurate DNA polymerases, with misinsertion and base substitution error rates when copying undamaged templates that generally range from 10–1 to 10–3 (Fig. 1, top). The most striking violation of Watson-Crick base pairing rules is exhibited by Pol ι, which inserts dGTP opposite template T even more efficiently than it inserts A opposite T (12Tissier A. McDonald J.P. Frank E.G. Woodgate R. Genes Dev. 2000; 14: 1642-1650PubMed Google Scholar, 13Zhang Y. Yuan F. Wu X. Wang Z. Mol. Cell. Biol. 2000; 20: 7099-7108Crossref PubMed Scopus (189) Google Scholar, 14Johnson R.E. Washington M.T. Haracska L. Prakash S. Prakash L. Nature. 2000; 406: 1015-1019Crossref PubMed Scopus (578) Google Scholar), i.e. its error rate for this mispair approaches 1 (Fig. 1). That base substitution error rates of wild-type DNA polymerases vary over a million-fold range is perhaps the biggest change in our view of DNA synthesis fidelity in the past decade. Crystal structures (Refs. 15Kunkel T.A. Bebenek K. Annu. Rev. Biochem. 2000; 69: 497-529Crossref PubMed Scopus (804) Google Scholar, 16Beard W.A. Wilson S.H. Chem. Biol. 1998; 5: R7-R13Abstract Full Text PDF PubMed Google Scholar, 17Steitz T.A. J. Biol. Chem. 1999; 274: 17395-17398Abstract Full Text Full Text PDF PubMed Scopus (699) Google Scholar, 18Doublié S. Sawaya M.R. Ellenberger T. Structure. 1999; 7: R31-R35Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 19Beard W.A. Wilson S.H. Structure. 2003; 11: 489-496Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 20Friedberg E.C. Fischhaber P.L. Kisker C. Cell. 2001; 107: 9-12Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 21Beard W.A. Wilson S.H. Structure. 2001; 9: 759-764Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 22Garcia-Diaz M. Bebenek K. Krahn J.M. Blanco L. Kunkel T.A. Pedersen L. Mol. Cell. 2004; 13: 561-572Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar and references therein) reveal that polymerase binding to DNA strongly reshapes the primer-template, e.g. the backbone at the templating base can be bent by 90°. In the absence of a dNTP, polymerases are often, but not invariably (20Friedberg E.C. Fischhaber P.L. Kisker C. Cell. 2001; 107: 9-12Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 21Beard W.A. Wilson S.H. Structure. 2001; 9: 759-764Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 22Garcia-Diaz M. Bebenek K. Krahn J.M. Blanco L. Kunkel T.A. Pedersen L. Mol. Cell. 2004; 13: 561-572Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), in an "open" conformation with the active site not yet assembled. Binding of a correct dNTP induces large changes in the relative positions of polymerase subdomains and more subtle changes in amino acid side chains and in DNA conformation. These dNTP-induced changes result is a "closed" ternary complex containing a binding pocket that snugly surrounds the nascent base pair and an active site containing the αP of the incoming dNTP poised for the in-line nucleophilic attack of the 3′-OH of the primer. Ternary complexes of several different polymerases have an arrangement of reactive groups consistent with a two-metal ion mechanism for nucleotidyl transfer that may be common to all polymerases (23Steitz T.A. Smerdon S.J. Jager J. Joyce C.M. Science. 1994; 266: 2022-2025Crossref PubMed Scopus (271) Google Scholar). Within this framework, the following ideas have been considered most relevant to fidelity. Ever since Watson and Crick (1Watson J.D. Crick F.H.C. Nature. 1953; 171: 737-738Crossref PubMed Scopus (8554) Google Scholar) noted that correct base pairs form specific hydrogen bonds, these have been thought to contribute to the specificity of DNA synthesis. That base-base hydrogen bonding does contribute to fidelity is clear, but the contribution appears to be relatively small and may be polymerase-dependent (24Kool E.T. Annu. Rev. Biochem. 2002; 71: 191-219Crossref PubMed Scopus (338) Google Scholar). By the late 1970s (25Loeb L.A. Kunkel T.A. Annu. Rev. Biochem. 1982; 51: 429-457Crossref PubMed Scopus (368) Google Scholar), the idea had emerged that if DNA polymerases merely acted as "zippers" to polymerize those dNTPs whose presence in the active site was determined by base-base hydrogen bonding, selectivity should depend on differences in free energy between complementary and non-complementary base pairs. In aqueous solution, these differences are 0.2–4 kcal/mol, which can account for one incorrect insertion for about 10 to a few hundred correct insertions. Error rates are in this range for Y family members (Fig. 1, top), suggesting that TLS enzymes may have relaxed geometric selectivity (see below) and primarily depend on base-base hydrogen bonding as the major determinant of fidelity. These ideas are supported by structural information (20Friedberg E.C. Fischhaber P.L. Kisker C. Cell. 2001; 107: 9-12Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 21Beard W.A. Wilson S.H. Structure. 2001; 9: 759-764Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 26Boudsocq F. Ling H. Yang W. Woodgate R. DNA Repair. 2002; 1: 343-358Crossref PubMed Scopus (73) Google Scholar, 27Ling H. Boudsocq F. Woodgate R. Yang W. Cell. 2001; 107: 91-102Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar, 28Ling H. Boudsocq F. Plosky B.S. Woodgate R. Yang W. Nature. 2003; 424: 1083-1087Crossref PubMed Scopus (202) Google Scholar) and by a report (29Washington M.T. Helquist S.A. Kool E.T. Prakash L. Prakash S. Mol. Cell. Biol. 2003; 23: 5107-5112Crossref PubMed Scopus (80) Google Scholar) that the insertion fidelity of yeast Pol η is severely impaired with difluorotoluene, a nonpolar isosteric analog of thymine that is unable to form Watson-Crick hydrogen bonds with adenine. Most polymerases have higher fidelity than can be explained by free energy differences between correct and incorrect base pairs in aqueous solution. One explanation (30Petruska J. Goodman M.F. Boosalis M.S. Sowers L.C. Cheong C. Tinoco Jr., I. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6252-6256Crossref PubMed Scopus (289) Google Scholar) is that these enzymes amplify free energy differences between correct and incorrect base pairs by partially excluding water from the active site, thus increasing enthalpy differences and reducing entropy differences and improving fidelity. This hypothesis is consistent with the observation that in the crystal structure of the Y family, low fidelity Sso Dpo4 (27Ling H. Boudsocq F. Woodgate R. Yang W. Cell. 2001; 107: 91-102Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar, 28Ling H. Boudsocq F. Plosky B.S. Woodgate R. Yang W. Nature. 2003; 424: 1083-1087Crossref PubMed Scopus (202) Google Scholar), the active site (Fig. 2, B and C) is more accessible to solvent than are the active sites of more accurate polymerases in other families (e.g. Pol β) (Fig. 2A). Polymerases in families A, B, X, and RT have nascent base pair binding pockets that tightly accommodate a correct Watson-Crick base pair (Fig. 2A and additional images in Refs. 15Kunkel T.A. Bebenek K. Annu. Rev. Biochem. 2000; 69: 497-529Crossref PubMed Scopus (804) Google Scholar, 16Beard W.A. Wilson S.H. Chem. Biol. 1998; 5: R7-R13Abstract Full Text PDF PubMed Google Scholar, 17Steitz T.A. J. Biol. Chem. 1999; 274: 17395-17398Abstract Full Text Full Text PDF PubMed Scopus (699) Google Scholar, 18Doublié S. Sawaya M.R. Ellenberger T. Structure. 1999; 7: R31-R35Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 19Beard W.A. Wilson S.H. Structure. 2003; 11: 489-496Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar and references therein). This tight fit is consistent with a concept that emerged about 25 years ago (reviewed in Refs. 15Kunkel T.A. Bebenek K. Annu. Rev. Biochem. 2000; 69: 497-529Crossref PubMed Scopus (804) Google Scholar and 31Echols H. Goodman M.F. Annu. Rev. Biochem. 1991; 60: 477-511Crossref PubMed Scopus (620) Google Scholar) that nucleotide selectivity largely depends on geometric selection for the shape and size of correct Watson-Crick base pairs. The geometries of A·T and G·C base pairs are remarkably similar to each other but differ from mismatched base pairs (15Kunkel T.A. Bebenek K. Annu. Rev. Biochem. 2000; 69: 497-529Crossref PubMed Scopus (804) Google Scholar, 19Beard W.A. Wilson S.H. Structure. 2003; 11: 489-496Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 24Kool E.T. Annu. Rev. Biochem. 2002; 71: 191-219Crossref PubMed Scopus (338) Google Scholar, 31Echols H. Goodman M.F. Annu. Rev. Biochem. 1991; 60: 477-511Crossref PubMed Scopus (620) Google Scholar, 32Kool E.T. Biopolymers. 1998; 48: 3-17Crossref PubMed Scopus (114) Google Scholar). Abnormal geometry is thought to result in steric clashes in and around the active site that preclude efficient catalysis. This hypothesis is supported by numerous studies with base analogs (24Kool E.T. Annu. Rev. Biochem. 2002; 71: 191-219Crossref PubMed Scopus (338) Google Scholar, 32Kool E.T. Biopolymers. 1998; 48: 3-17Crossref PubMed Scopus (114) Google Scholar). As one example, nonpolar bases that mimic the size and shape of normal bases but are unable to form Watson-Crick hydrogen bonds are incorporated by some polymerases with selectivity almost as high as for normal bases, suggesting that base pair shape and size may contribute more to the fidelity of some accurate DNA polymerases than does base-base hydrogen bonding. Small pyrimidine-pyrimidine mispairs that might otherwise fit into the binding pocket may be enlarged by water molecules that hydrogen bond to their Watson-Crick pairing edges. This effect of solvation is suggested to provide a strong force for steric exclusion (32Kool E.T. Biopolymers. 1998; 48: 3-17Crossref PubMed Scopus (114) Google Scholar). The importance of geometry to fidelity is also implied by the altered fidelity of polymerases with non-conservative replacements of amino acids that are in and adjacent to the polymerase active site (e.g. see Refs. 15Kunkel T.A. Bebenek K. Annu. Rev. Biochem. 2000; 69: 497-529Crossref PubMed Scopus (804) Google Scholar and 24Kool E.T. Annu. Rev. Biochem. 2002; 71: 191-219Crossref PubMed Scopus (338) Google Scholar). Polymerase interactions important to fidelity occur with the minor groove edges of the templating nucleotide and the primer-terminal base pair (e.g. magenta patch in Fig. 2A), with the base, sugar, and triphosphate moieties of the incoming nucleotide, and with the template strand nucleotide immediately 5′ to the templating nucleotide. Altered fidelity also results from changing amino acids that interact several base pairs upstream in the as as amino acids that not the DNA or The changes may geometry or in the not in crystal The importance of size and shape is also implied by the generally low fidelity of Y family polymerases and their to copy DNA templates containing lesions that In the crystal structure of a ternary complex of Sso Dpo4 (27Ling H. Boudsocq F. Woodgate R. Yang W. Cell. 2001; 107: 91-102Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar) with DNA and an active site (Fig. of small side chains and to accommodate bases at the (Fig. This may be for lesions that studies (reviewed in Refs. 15Kunkel T.A. Bebenek K. Annu. Rev. Biochem. 2000; 69: 497-529Crossref PubMed Scopus (804) Google Scholar, 19Beard W.A. Wilson S.H. Structure. 2003; 11: 489-496Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, M.F. S. J. Rev. Biochem. Mol. Biol. 1993; PubMed Scopus Google Scholar, Annu. Rev. Biochem. 1993; PubMed Scopus Google Scholar, 2002; PubMed Scopus Google Scholar, W.A. Wilson S.H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) have that fidelity depends on differences in the binding and insertion rates of incorrect correct The that incorrect and correct nucleotide insertion an active of and may depend on the the base and the DNA sequence is generally that nucleotide insertion is by an dNTP-induced change A recent W.A. Wilson S.H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) that the for correct dNTP insertion by different polymerases by with the least accurate polymerases the In for incorrect by these polymerases vary over a The for correct insertion are due to the different insertion rates exhibited by DNA polymerases because generally the correct dNTP with similar W.A. Wilson S.H. Structure. 2003; 11: 489-496Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, W.A. Wilson S.H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). A of structures that the of the of the incoming correct dNTP polymerases with different W.A. Wilson S.H. Structure. 2003; 11: 489-496Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). These results suggest that fidelity is primarily by the ability to the correct nucleotide W.A. Wilson S.H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) and on the changes and interactions to and the The dNTP-induced to change from was considered as a possible but recent studies W.A. Wilson S.H. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, W. X. J. Z. C. 2001; PubMed Scopus Google Scholar, L. W.A. Wilson S.H. S. T. J. Mol. Biol. 2002; Scopus Google Scholar) suggest that this may not be the for DNA polymerase Pol has similar base substitution fidelity to Pol yet Pol it is in a conformation even a dNTP M. Bebenek K. Krahn J.M. Blanco L. Kunkel T.A. Pedersen L. Mol. Cell. 2004; 13: 561-572Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Y family polymerases are suggested to be in a conformation dNTP (Refs. 20Friedberg E.C. Fischhaber P.L. Kisker C. Cell. 2001; 107: 9-12Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar and 21Beard W.A. Wilson S.H. Structure. 2001; 9: 759-764Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar but also see in 28Ling H. Boudsocq F. Plosky B.S. Woodgate R. Yang W. Nature. 2003; 424: 1083-1087Crossref PubMed Scopus (202) Google Scholar), yet M.T. Prakash L. Prakash S. Cell. 2001; 107: Full Text Full Text PDF PubMed Scopus Google Scholar) have the importance of a dNTP-induced change to the fidelity of Pol These suggest that fidelity may be by subtle changes that result in of active site and that these may differ for correct and incorrect a recent Joyce C.M. 2003; PubMed Scopus Google Scholar) has suggested that the during correct and incorrect nucleotide are and that specificity for the correct be if these differences into the consistent with an fit mechanism see Refs. Joyce C.M. 2003; PubMed Scopus Google Scholar and Jr., PubMed Scopus Google Scholar). When copying undamaged polymerases base substitution at that mispairs can with the stability and geometry for catalysis. These may be minor forms from base pairing (e.g. L. Kunkel T.A. Joyce Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar), or of bases M.F. S. J. Rev. Biochem. Mol. Biol. 1993; PubMed Scopus Google Scholar). The most known of the is synthesis involving a common of many polymerases, in a conformation forms a pair with generating mutations (reviewed in Refs. M. 1993; 9: Full Text PDF PubMed Scopus Google Scholar and R.M. Rev. PubMed Google Scholar). Pol this by the backbone of the templating nucleotide a steric with the at and pairing of incoming with in the Watson-Crick conformation J.M. W.A. H. Wilson S.H. Structure. 2003; 11: Full Text Full Text PDF PubMed Scopus Google Scholar). a of base pairing is for Sso Dpo4 of a In a ternary crystal structure complex H. Boudsocq F. Plosky B.S. Woodgate R. Yang W. Nature. 2003; 424: 1083-1087Crossref PubMed Scopus (202) Google Scholar), the T of the forms a Watson-Crick base pair with in the conformation at the active site the 5′ T forms a base pair with in its conformation. base pairing is one possible to correct templating by the 5′ T its to the This may have for the fidelity of because Dpo4 and human Pol η have higher fidelity at the 5′ T of a than at the and the fidelity of human Pol η is higher at the 5′ T of a than at the undamaged T C. S. F. Kunkel T.A. Nature. 2004; PubMed Scopus Google Scholar). These different for DNA synthesis with that have studies have the nucleotide insertion specificity of polymerases opposite other lesions (Refs. 8Kunkel T.A. Pavlov Y.I. Benenek K. DNA Repair. 2003; 2: 135-149Crossref PubMed Scopus (118) Google Scholar, 10Prakash S. Prakash L. Genes Dev. 2002; 16: 1872-1883Crossref PubMed Scopus (295) Google Scholar, 11Livneh Z. J. Biol. Chem. 2001; 276: 25639-25642Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, A. McDonald J.P. Frank E.G. Woodgate R. Genes Dev. 2000; 14: 1642-1650PubMed Google Scholar, 13Zhang Y. Yuan F. Wu X. Wang Z. Mol. Cell. Biol. 2000; 20: 7099-7108Crossref PubMed Scopus (189) Google Scholar, 14Johnson R.E. Washington M.T. Haracska L. Prakash S. Prakash L. Nature. 2000; 406: 1015-1019Crossref PubMed Scopus (578) Google Scholar, H. Wang Z. Frank E.G. H. F. M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, S. C. S.A. Chem. Res. 2003; 16: PubMed Scopus Google Scholar and references therein). the of polymerases and the large number of lesions by cellular metabolism and external environmental the for with other to fully damage-induced replication DNA polymerases mismatched less efficiently than matched primer. This is that in-line nucleophilic attack of the 3′-OH of the and that the nascent base pair binding pocket is by the primer-terminal base pair (Fig. The of is M.F. S. J. Rev. Biochem. Mol. Biol. 1993; PubMed Scopus Google Scholar), with mismatched (e.g. more than others (e.g. polymerases Pol and Pol are to be at mismatch S. Prakash L. Genes Dev. 2002; 16: 1872-1883Crossref PubMed Scopus (295) Google Scholar), which may their roles in following opposite a by polymerase S. Prakash L. Genes Dev. 2002; 16: 1872-1883Crossref PubMed Scopus (295) Google Scholar). polymerases between correct and incorrect by M.F. S. J. Rev. Biochem. Mol. Biol. 1993; PubMed Scopus Google Scholar), the to polymerases with intrinsic proofreading activity, mismatch the to and to the exonuclease active site to and the balance between mismatch and excision the contribution of proofreading to fidelity (15Kunkel T.A. Bebenek K. Annu. Rev. Biochem. 2000; 69: 497-529Crossref PubMed Scopus (804) Google Scholar, M.F. S. J. Rev. Biochem. Mol. Biol. 1993; PubMed Scopus Google Scholar, Annu. Rev. Biochem. 1993; PubMed Scopus Google Scholar). This balance can be by amino acid replacements at either of the active sites or even between them in 15Kunkel T.A. Bebenek K. Annu. Rev. Biochem. 2000; 69: 497-529Crossref PubMed Scopus (804) Google Scholar) or by increasing the dNTP to in of and structural see Refs. 15Kunkel T.A. Bebenek K. Annu. Rev. Biochem. 2000; 69: 497-529Crossref PubMed Scopus (804) Google Scholar, M.F. S. J. Rev. Biochem. Mol. Biol. 1993; PubMed Scopus Google Scholar, and Annu. Rev. Biochem. 1993; PubMed Scopus Google Scholar). many (e.g. at least 10 of polymerases lack intrinsic proofreading (2Hübscher U. Maga G. Spadari S. Annu. Rev. Biochem. 2002; 71: 133-163Crossref PubMed Scopus (588) Google Scholar, 3Shcherbakova P.V. Bebenek K. Kunkel T.A. Sci. Aging Knowl. 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Biol. 2000; PubMed Scopus Google Scholar). base can also result from misinsertion of a base followed by to the mismatched into a matched with an template base in the upstream may also be by in the active site (e.g. see Fig. when an incoming dNTP forms a correct Watson-Crick base pair but with the template and active site can result in with one or more correct base more efficient following insertion of an incorrect nucleotide or insertion opposite a (Refs. 15Kunkel T.A. Bebenek K. Annu. Rev. Biochem. 2000; 69: 497-529Crossref PubMed Scopus (804) Google Scholar, T.A. PubMed Scopus Google Scholar, K. Kunkel T.A. Biol. 2000; PubMed Scopus Google Scholar, Science. 2003; PubMed Scopus Google Scholar and references therein). can also substitution at high rates by a in which is followed by correct insertion and and mismatch T.A. PubMed Scopus Google Scholar). When strand occur in the can be at some from the polymerase active site and the can be by correct base pairs whose number with increasing sequence G. Y. J. J. A. E. M. Biol. PubMed Scopus Google Scholar). Thus, long can provide that over proofreading. This the error rates of most polymerases with increasing sequence and even replicative DNA polymerases with high base substitution fidelity have low base fidelity (e.g. Fig. 1, when copying long (e.g. Refs. G. Y. J. J. A. E. M. Biol. PubMed Scopus Google Scholar and D. Kunkel T.A. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar and references therein). That such low fidelity during replication in vivo is by the high of sequence in cells lacking the ability to correct replication by mismatch are not to small of DNA synthesis between and DNA polymerases can also and complex involving of A more of insertion and during DNA synthesis can be found in Refs. G. Y. J. J. A. E. M. Biol. PubMed Scopus Google Scholar and T.A. PubMed Scopus Google Scholar with structural perspectives in 15Kunkel T.A. Bebenek K. Annu. Rev. Biochem. 2000; 69: 497-529Crossref PubMed Scopus (804) Google A of replication fidelity for should an of the between DNA sequence and of neurodegenerative and at in Bebenek and A. for several great and of this