Evidence for Extrinsic Exonucleolytic Proofreading

McElhinny, Stephanie A. Nick; Pavlov, Youri I.; Kunkel, Thomas A.

doi:10.4161/cc.5.9.2736

Cited by 45 publications

(44 citation statements)

References 35 publications

(47 reference statements)

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“…Thus, V522A suppresses the most prevalent mutations generated by proofreadingdeficient Pol e to a similar degree. This pattern of suppression is consistent with a mutant polymerase that exhibits either increased overall nucleotide selectivity or increased facility to undergo extrinsic proofreading (Albertson and Preston 2006;Nick McElhinny et al 2006;Pavlov et al 2006a). The G435C antimutator maps to Pol d R475 in a loop of the exonuclease domain that extends into and interacts with the thumb domain ( Figure 7, A and F).…”

Section: Palombosupporting

confidence: 49%

Emergence of DNA Polymerase ε Antimutators That Escape Error-Induced Extinction in Yeast

2013

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DNA polymerases (Pols) e and d perform the bulk of yeast leading-and lagging-strand DNA synthesis. Both Pols possess intrinsic proofreading exonucleases that edit errors during polymerization. Rare errors that elude proofreading are extended into duplex DNA and excised by the mismatch repair (MMR) system. Strains that lack Pol proofreading or MMR exhibit a 10-to 100-fold increase in spontaneous mutation rate (mutator phenotype), and inactivation of both Pol d proofreading (pol3-01) and MMR is lethal due to replication error-induced extinction (EEX). It is unclear whether a similar synthetic lethal relationship exists between defects in Pol e proofreading (pol2-4) and MMR. Using a plasmid-shuffling strategy in haploid Saccharomyces cerevisiae, we observed synthetic lethality of pol2-4 with alleles that completely abrogate MMR (msh2D, mlh1D, msh3D msh6D, or pms1D mlh3D) but not with partial MMR loss (msh3D, msh6D, pms1D, or mlh3D), indicating that high levels of unrepaired Pol e errors drive extinction. However, variants that escape this error-induced extinction (eex mutants) frequently emerged. Five percent of pol2-4 msh2D eex mutants encoded second-site changes in Pol e that reduced the pol2-4 mutator phenotype between 3-and 23-fold. The remaining eex alleles were extragenic to pol2-4. The locations of antimutator amino-acid changes in Pol e and their effects on mutation spectra suggest multiple mechanisms of mutator suppression. Our data indicate that unrepaired leading-and lagging-strand polymerase errors drive extinction within a few cell divisions and suggest that there are polymerase-specific pathways of mutator suppression. The prevalence of suppressors extragenic to the Pol e gene suggests that factors in addition to proofreading and MMR influence leading-strand DNA replication fidelity.O RGANISMS must accurately duplicate their genomes to avoid loss of long-term fitness. Consequently, cells employ high-fidelity DNA polymerases (Pols) equipped with proofreading exonucleases to replicate their DNA (reviewed in McCulloch and Kunkel 2008;Reha-Krantz 2010). Mismatch repair (MMR) further ensures the integrity of genetic information by targeting mismatches for excision from newly replicated DNA (reviewed in Kolodner and Marsischky 1999;Iyer et al. 2006;Hsieh and Yamane 2008). These polymerase error-correcting mechanisms, together with DNA damage repair (Friedberg et al. 2006), maintain the genome with less than one mutation per 10 9 nucleotides per cell division (Drake et al. 1998). Defects in proofreading or MMR result in mutator phenotypes characterized by increased rates of spontaneous mutation (Kolodner and Marsischky 1999;Iyer et al. 2006;Hsieh and Yamane 2008;McCulloch and Kunkel 2008; RehaKrantz 2010).Mutator phenotypes can be an important source of genetic diversity, which facilitates adaptation to environmental change. In bacterial and yeast populations, unstable environments favor mutator strains that readily acquire adaptive mutations (Chao and Cox 1983;Mao et al. 1997;Sniegowski et al. 1997...

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Section: Palombosupporting

confidence: 49%

Emergence of DNA Polymerase ε Antimutators That Escape Error-Induced Extinction in Yeast

2013

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“…This is a substantial workload for a polymerase that cannot proofread its own errors and has an average error rate of ∼10 −4 (40), which is increased approximately fivefold by the L868M substitution (23). To maintain genome stability when initiating ∼50,000 Okazaki fragments during replication of the yeast genome, Pol δ (but not Pol ε) has been suggested to "extrinsically" proofread errors made by Pol α (23,41). Extrinsic proofreading may occur largely after Pol α incorporates enough deoxynucleotides (e.g., 5-10 nucleotides) to provide Pol δ with a fully DNA-DNA duplex, as Pol δ may not function well nearer to the 5′ end, where an RNA-DNA duplex initially exists (Fig.…”

Section: Discussionmentioning

confidence: 99%

Differential correction of lagging-strand replication errors made by DNA polymerases α and δ

McElhinny

Kissling

Kunkel

2010

Proc. Natl. Acad. Sci. U.S.A.

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Mismatch repair (MMR) of replication errors requires DNA ends that can direct repair to the newly synthesized strand containing the error. For all but those organisms that use adenine methylation to generate nicks, the source of these ends in vivo is unknown. One possibility is that MMR may have a "special relation to the replication complex" [Wagner R, Jr., Meselson M (1976) Proc Natl Acad Sci USA 73: [4135][4136][4137][4138][4139], perhaps one that allows 5′ or 3′ DNA ends associated with replication to act as strand discrimination signals. Here we examine this hypothesis, based on the logic that errors made by yeast DNA polymerase α (Pol α), which initiates Okazaki fragments during lagging-strand replication, will always be closer to a 5′ end than will be more internal errors generated by DNA polymerase δ (Pol δ), which takes over for Pol α to complete lagging-strand replication. When we compared MMR efficiency for errors made by variant forms of these two polymerases, Msh2-dependent repair efficiencies for mismatches made by Pol α were consistently higher than for those same mismatches when made by Pol δ. Thus, one special relationship between MMR and replication is that MMR is more efficient for the least accurate of the major replicative polymerases, exonuclease-deficient Pol α. This observation is consistent with the close proximity and possible use of 5′ ends of Okazaki fragments for strand discrimination, which could increase the probability of Msh2-dependent MMR by 5′ excision, by a Msh2-dependent strand displacement mechanism, or both.replication fork | DNA repair | infidelity | mutagenesis | base substitutions V arious types of DNA repair, including base excision repair, nucleotide excision repair, double-strand DNA break repair, and mismatch repair (MMR), are differentiated into subpathways that can be distinguished by substrate specificity and enzymology (1). This is particularly true for DNA mismatch repair (reviewed in refs. 2-6), which can involve any of several different protein complexes at sequential steps in the pathway. For example, repair of mismatches generated during eukaryotic DNA replication is initiated upon mismatch binding by Msh2-Msh6 or Msh2-Msh3, and subsequent steps can involve three different MutL heterodimers (reviewed in ref. 5). Starting from a nick in the nascent DNA strand, mismatches can be removed by excision. Recent evidence suggests that mismatches may also sometimes be removed by a strand displacement mechanism (7). In either case, removal of the mismatch requires a DNA strand discontinuity. Studies in vitro demonstrate that this discontinuity can be a nick located either 3′ or 5′ to the mismatch, with the protein requirements for MMR differing somewhat depending on the location of the nick relative to the mismatch (see refs. 2 and 7 and references therein).Early experiments in Escherichia coli revealed that one signal that directs MMR to the nascent strand is transient under-methylation of adenine in GATC sequences, allowing MutH endonuclease to nick that strand to d...

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“…However, both the human XPF-ERCC1 complex (38) and the yeast homologue, Rad1-Rad10 complex (37), have a 3Ј-to 5Ј-exonuclease activity. In addition, it has been suggested that pol ␦ may provide such activity during the course of ICL removal (45). Importantly, most proteins employed by this proposed mechanism are associated with the nucleotide excision repair machinery, including pol that was shown to function in this pathway (46 …”

Section: Discussionmentioning

confidence: 99%

Role for DNA Polymerase κ in the Processing of N2-N2-Guanine Interstrand Cross-links

Minko

Harbut

Kozekov

et al. 2008

Journal of Biological Chemistry

121

187

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Although there exists compelling genetic evidence for a homologous recombination-independent pathway for repair of interstrand cross-links (ICLs) involving translesion synthesis (TLS), biochemical support for this model is lacking. To identify DNA polymerases that may function in TLS past ICLs, oligodeoxynucleotides were synthesized containing site-specific ICLs in which the linkage was between N 2 -guanines, similar to crosslinks formed by mitomycin C and enals. Here, data are presented that mammalian cell replication of DNAs containing these lesions was ϳ97% accurate. Using a series of oligodeoxynucleotides that mimic potential intermediates in ICL repair, we demonstrate that human polymerase (pol) not only catalyzed accurate incorporation opposite the cross-linked guanine but also replicated beyond the lesion, thus providing the first biochemical evidence for TLS past an ICL. The efficiency of TLS was greatly enhanced by truncation of both the 5 and 3 ends of the nontemplating strand. Further analyses showed that although yeast Rev1 could incorporate a dCTP opposite the cross-linked guanine, no evidence was found for TLS by pol or a pol /Rev1 combination. Because pol was able to bypass these ICLs, biological evidence for a role for pol in tolerating the N 2 -N 2 -guanine ICLs was sought; both cell survival and chromosomal stability were adversely affected in pol -depleted cells following mitomycin C exposure. Thus, biochemical data and cellular studies both suggest a role for pol in the processing of N 2 -N 2 -guanine ICLs.

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Evidence for Extrinsic Exonucleolytic Proofreading

Cited by 45 publications

References 35 publications

Emergence of DNA Polymerase ε Antimutators That Escape Error-Induced Extinction in Yeast

Emergence of DNA Polymerase ε Antimutators That Escape Error-Induced Extinction in Yeast

Differential correction of lagging-strand replication errors made by DNA polymerases α and δ

Role for DNA Polymerase κ in the Processing of N2-N2-Guanine Interstrand Cross-links

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