DNA Replication Fidelity: Proofreading in Trans

Albertson, Tina; Preston, Bradley D.

doi:10.1016/j.cub.2006.02.031

Cited by 22 publications

(20 citation statements)

References 21 publications

(46 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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: Escape From Pol E Error-induced Extinctionsupporting

confidence: 70%

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

2013

View full text Add to dashboard Cite

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...

show abstract

Section: Escape From Pol E Error-induced Extinctionsupporting

confidence: 70%

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

2013

View full text Add to dashboard Cite

show abstract

“…fivefold increases in the spontaneous mutation rate at the Hprt locus (Table 1) may reflect both defective synthesis by the mutant enzymes, encompassing reduced base discrimination, catalytic efficiency, and/or processivity, as well as secondary mutagenic processes resulting from flawed replication. It is likely that the polymerase active site in the mutant enzymes generates more errors than the measured mutation rates indicate, due to proofreading at the exonuclease active site of Pol ␦ and other DNA polymerases, and to error correction by mismatch repair and perhaps additional exonucleases (1,27,56). We also observed 17-and 38-fold increases in chromosome anomalies in heterozygous L604G and L604K cells, respectively, due primarily to chromatid/chromosome breaks and gaps (Fig.…”

Section: Discussionmentioning

confidence: 71%

Mutation at the Polymerase Active Site of Mouse DNA Polymerase δ Increases Genomic Instability and Accelerates Tumorigenesis

Venkatesan¹,

Treuting

Fuller³

et al. 2007

Molecular and Cellular Biology

Self Cite

View full text Add to dashboard Cite

Mammalian DNA polymerase ␦ (Pol ␦) is believed to replicate a large portion of the genome and to synthesize DNA in DNA repair and genetic recombination pathways. The effects of mutation in the polymerase domain of this essential enzyme are unknown. Here, we generated mice harboring an L604G or L604K substitution in highly conserved motif A in the polymerase active site of Pol ␦. Homozygous Pold1 L604G/L604G and Pold1 L604K/L604K mice died in utero. However, heterozygous animals were viable and displayed no overall increase in disease incidence, indicative of efficient compensation for the defective mutant polymerase. The life spans of wild-type and heterozygous Pold1 ؉/L604G mice did not differ, while that of Pold1 ؉/L604K mice was reduced by 18%. Cultured embryonic fibroblasts from the heterozygous strains exhibited comparable increases in both spontaneous mutation rate and chromosome aberrations. We observed no significant increase in cancer incidence; however, Pold1 ؉/L604K mice bearing histologically diagnosed tumors died at a younger median age than wild-type mice. Our results indicate that heterozygous mutation at L604 in the polymerase active site of DNA polymerase ␦ reduces life span, increases genomic instability, and accelerates tumorigenesis in an allele-specific manner, novel findings that have implications for human cancer.Eukaryotic DNA polymerase ␦ (Pol ␦) is an essential, highly conserved enzyme that participates in DNA replication, DNA repair, and genetic recombination. Pol ␦ is believed to replicate a large portion of the genome, synthesizing most of the lagging strand and perhaps contributing to leading-strand synthesis as well (14, 22, 43) The 125-kDa catalytic subunit, encoded at the mouse Pold1 locus, contains a polymerase domain near the carboxyl terminus that catalyzes DNA synthesis and an exonuclease domain near the amino terminus that catalyzes 3Ј35Ј exonucleolytic proofreading (5, 18). Pol ␦ is highly processive in association with PCNA and synthesizes DNA with great accuracy, catalyzing about one error in 10 Ϫ5 to 10 Ϫ6 nucleotides polymerized (11,46,61). Discrimination between correct and incorrect base pairs at the polymerase active site confers most of the fidelity (error rate, ca.

show abstract

“…Similarities to the proofreading exonucleases and the preference for frayed DNA ends lead to the proposal that the TREX enzymes might serve an extrinsic function to proofread errors generated by DNA polymerases that lack intrinsic exonuclease activities (5, 6). There is some genetic evidence for extrinsic proofreading by some exonucleases of DNA polymerases (35)(36)(37), but evidence that the TREX enzymes act as proofreaders is supported only by in vitro studies (1). Although direct genetic evidence for a TREX2 role in mammalian DNA maintenance is lacking, cells deleted of TREX2 exhibit increased levels of chromosomal rearrangements suggesting a role in genome stability (38).…”

mentioning

confidence: 99%

Cooperative DNA Binding and Communication across the Dimer Interface in the TREX2 3′ → 5′-Exonuclease

Perrino

Silva

Harvey

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

The activity of human TREX2-catalyzed 3 3 5-deoxyribonuclease has been analyzed in steady-state and single turnover kinetic assays and in equilibrium DNA binding studies. These kinetic data provide evidence for cooperative DNA binding within TREX2 and for coordinated catalysis between the TREX2 active sites supporting a model for communication between the protomers of a TREX2 dimer. Mobile loops positioned adjacent to the active sites provide the major DNA binding contribution and facilitate subsequent binding into the active sites. Mutations of three arginine residues on these loops cause decreased TREX2 activities by up to 60-fold. Steady-state kinetic assays of these arginine to alanine TREX2 variants result in increased K m values for DNA substrate with no effect on k cat values indicating contributions exclusively to DNA binding by all three of the loop arginines. TREX2 heterodimers were prepared to determine whether exonuclease activity in one protomer is communicated to the opposing protomer. Evidence for communication across the dimer interface is provided by the 7-fold lower catalytic activity measured in the TREX2 WT/H188A heterodimer compared with the TREX2 WT homodimer, contrasting the 2-fold lower activity measured in the TREX2 WT/R163A,R165A,R167A heterodimer. The measured activity in TREX2WT/H188A heterodimer indicates that defective catalysis in one protomer reduces activity in the opposing protomer. A DNA binding analysis of TREX2 and the heterodimers indicates a cooperative binding effect within the TREX2 protomer. Finally, single turnover kinetic assays identify DNA binding as the rate-limiting step in TREX2 catalysis.The 3Ј 3 5Ј-deoxyribonucleases play fundamental roles in a variety of DNA metabolic pathways. These enzymes process DNA 3Ј ends by removing nucleotides one at a time to remodel 3Ј termini for subsequent molecular events in cells (1, 2). TREX1 is the most active 3Ј-deoxyribonuclease purified from mammalian cells (3, 4). TREX2 has not been purified from cells, but recombinant TREX1 and TREX2 generated in Escherichia coli confirm the robust nature of these deoxyribonucleases with TREX1 exhibiting about a 10-fold greater activity than TREX2(5-7). The high catalytic efficiencies measured for TREX1 and TREX2 can be attributed in part to the nature of the active sites. The TREX gene sequences and Mg 2ϩ ion dependence suggested that these deoxyribonucleases might be members of the DnaQ-like exonuclease family that utilize a two Mg 2ϩ ion-dependent mechanism for catalysis (8 -12). The DnaQ-like family contains both deoxyribo-and riboexonucleases and has been divided into two subfamilies characterized by the presence of four conserved carboxylate residues and a histidine (DEDD-h) or a tyrosine (DEDD-y) positioned in the active site (13). The TREX enzymes prefer excision of deoxynucleotide polymers over ribonucleotide polymers by about 1000-fold (5) 2 with specificity for unmodified 3Ј termini (14, 15). The four carboxylate residue side chains in the DnaQ-like enzymes coordinate two divalen...

show abstract

DNA Replication Fidelity: Proofreading in Trans

Abstract: Proofreading is the primary guardian of DNA polymerase fidelity. New work has revealed that polymerases with intrinsic proofreading activity may cooperate with non-proofreading polymerases to ensure faithful DNA replication.

Cited by 22 publications

References 21 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

Mutation at the Polymerase Active Site of Mouse DNA Polymerase δ Increases Genomic Instability and Accelerates Tumorigenesis

Cooperative DNA Binding and Communication across the Dimer Interface in the TREX2 3′ → 5′-Exonuclease

Contact Info

Product

Resources

About