Analysis of Damage Tolerance Pathways in <i>Saccharomyces cerevisiae</i>: a Requirement for Rev3 DNA Polymerase in Translesion Synthesis

Baynton, K.; Bresson-Roy, A.; Fuchs, Robert P. P.

doi:10.1128/mcb.18.2.960

Cited by 75 publications

(66 citation statements)

References 46 publications

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“…Although psoralen monoadducts are substrates for NER, a single lesion on an extrachromosomally replicating plasmid did not greatly reduce the measured transformation level, perhaps because of replication of the undamaged strand (36). As with repair-proficient cells, monoadducts did not induce plasmid integration or conversion to His ϩ .…”

Section: Resultsmentioning

confidence: 90%

DNA Repair Defects Channel Interstrand DNA Cross-links into Alternate Recombinational and Error-prone Repair Pathways

Saffran

Ahmed

Bellevue

et al. 2004

Journal of Biological Chemistry

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The repair of psoralen interstrand cross-links in the yeast Saccharomyces cerevisiae involves the DNA repair groups nucleotide excision repair (NER), homologous recombination (HR), and post-replication repair (PRR). In repair-proficient yeast cells cross-links induce double-strand breaks, in an NER-dependent process; the double-strand breaks are then repaired by HR. An alternate error-prone repair pathway generates mutations at cross-link sites. We have characterized the repair of plasmid molecules carrying a single psoralen cross-link, psoralen monoadduct, or double-strand break in yeast cells with deficiencies in NER, HR, or PRR genes, measuring the repair efficiencies and the levels of gene conversions, crossing over, and mutations. Strains with deficiencies in the NER genes RAD1, RAD3, RAD4, and RAD10 had low levels of cross-link-induced recombination but higher mutation frequencies than repair-proficient cells. Deletion of the HR genes RAD51, RAD52, RAD54, RAD55, and RAD57 also decreased induced recombination and increased mutation frequencies above those of NER-deficient yeast. Strains lacking the PRR genes RAD5, RAD6, and RAD18 did not have any crosslink-induced mutations but showed increased levels of recombination; rad5 and rad6 cells also had altered patterns of cross-link-induced gene conversion in comparison with repair-proficient yeast. Our observations suggest that psoralen cross-links can be repaired by three pathways: an error-free recombinational pathway requiring NER and HR and two PRR-dependent errorprone pathways, one NER-dependent and one NERindependent.DNA interstrand cross-links are complex lesions, highly toxic to cells, and difficult to repair because of the involvement of both strands of the DNA duplex. A single unrepaired interstrand cross-link is sufficient to kill a cell, and multiple DNA repair pathways may be required to complete cross-link removal (1-7).The yeast Saccharomyces cerevisiae has three major DNA repair epistasis groups, all of which are involved in cross-link repair; they are nucleotide excision repair, recombinational repair, and post-replication repair (8). Nucleotide excision repair (NER) 1 is a general system that recognizes bulky and helix-distorting lesions (9, 10). Endonucleases nick the affected DNA strand on both the 5Ј and 3Ј sides of the lesion; after removal of the damaged oligonucleotide, the resulting singlestrand gap is filled in by polymerase activity using the undamaged strand as a template. This mechanism produces error-free repair of single-strand damage. Recombinational repair is the major pathway for repair of double-strand damage, such as double-strand breaks (DSBs) or gaps in yeast (11,12). In homologous recombination (HR) the broken DNA molecule is repaired, and missing genetic information is restored through interactions with intact homologous sequences. This pathway is also non-mutagenic but can result in DNA rearrangements. Post-replication repair (PRR) acts on damage in the context of DNA replication, allowing for bypass of replication fork-...

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Section: Resultsmentioning

confidence: 90%

DNA Repair Defects Channel Interstrand DNA Cross-links into Alternate Recombinational and Error-prone Repair Pathways

Saffran

Ahmed

Bellevue

et al. 2004

Journal of Biological Chemistry

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“…In addition, we have carried out studies of TLS through a number of other DNA lesions-a cis-syn TT dimer, a (6-4) TT photoproduct, and an AAF adduct-that are also carried on a duplex plasmid. Also in this plasmid, replication initiates from a single-origin site and proceeds through a site-specific DNA lesion (Baynton et al 1998;Bresson and Fuchs 2002). From all these studies we conclude a requirement of Rad5 for TLS dependent upon Polz.…”

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confidence: 73%

Requirement of Rad5 for DNA Polymerase ζ-Dependent Translesion Synthesis in Saccharomyces cerevisiae

et al. 2008

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In yeast, Rad6-Rad18-dependent lesion bypass involves translesion synthesis (TLS) by DNA polymerases h or z or Rad5-dependent postreplication repair (PRR) in which error-free replication through the DNA lesion occurs by template switching. Rad5 functions in PRR via its two distinct activities-a ubiquitin ligase that promotes Mms2-Ubc13-mediated K63-linked polyubiquitination of PCNA at its lysine 164 residue and a DNA helicase that is specialized for replication fork regression. Both these activities are important for Rad5's ability to function in PRR. Here we provide evidence for the requirement of Rad5 in TLS mediated by Polz. Using duplex plasmids carrying different site-specific DNA lesions-an abasic site, a cis-syn TT dimer, a (6-4) TT photoproduct, or a G-AAF adduct-we show that Rad5 is needed for Polz-dependent TLS. Rad5 action in this role is likely to be structural, since neither the inactivation of its ubiquitin ligase activity nor the inactivation of its helicase activity impairs its role in TLS.

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“…It was suggested that MMR reduces the efficiency of recombination-mediated bypass when damage is present, and the studies presented here provide a concrete model for how the MMR system modulates pathway choice. Finally, our results may provide an explanation for the very low frequency (1-5%) of Pol -dependent lesion bypass observed in some plasmid-based yeast transformation studies (32,33), which contrasts with the high frequency (Ϸ60%) obtained when using lesion-containing singlestranded oligonucleotides (34,35). Based on the clear MMR dependence of TLS observed in the lys2⌬A746-NR system, we suggest that the specific use of MMR-defective host strains in the plasmid-based studies could account for the low TLS frequency.…”

Section: Discussionmentioning

confidence: 96%

The mismatch repair system promotes DNA polymerase ζ-dependent translesion synthesis in yeast

Lehner

Jinks-Robertson

2009

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

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DNA lesions that block replication can be bypassed by error-prone or error-free mechanisms. Error-prone mechanisms rely on specialized translesion synthesis (TLS) DNA polymerases that directly replicate over the lesion, whereas error-free pathways use an undamaged duplex as a template for lesion bypass. In the yeast Saccharomyces cerevisiae, most mutagenic TLS of spontaneous and induced DNA damage relies on DNA polymerase (Pol ) activity. Here, we use a distinct mutational signature produced by Pol in a frameshift-reversion assay to examine the role of the yeast mismatch repair (MMR) system in regulating Pol -dependent mutagenesis. Whereas MMR normally reduces mutagenesis by removing errors introduced by replicative DNA polymerases, we find that the MMR system is required for Pol -dependent mutagenesis. In the absence of homologous recombination, however, the errorprone Pol pathway is not affected by MMR status. These results demonstrate that MMR promotes Pol -dependent mutagenesis by inhibiting an alternative, error-free pathway that depends on homologous recombination. Finally, in contrast to its ability to remove mistakes made by replicative DNA polymerases, we show that MMR fails to efficiently correct errors introduced by Pol .DNA damage ͉ mutagenesis ͉ recombination ͉ replication M utations generally impair fitness and are important contributors to genome instability and disease, but a low level of mutagenesis is required to provide raw material for evolution. Spontaneous mutations result from endogenous metabolic processes and can be attributed either to errors made when copying an undamaged DNA template or to errors introduced when replicating over a DNA lesion. Mistakes of the first type are corrected by the proofreading activity of the replicative DNA polymerases or by the postreplicative mismatch repair (MMR) machinery (1, 2). In addition to monitoring replication fidelity (spellchecker function), the MMR system also monitors the fidelity of homologous recombination (3). By reducing interactions between sequences that are not identical, MMR-associated antirecombination activity limits genomic rearrangements between dispersed repeats. Finally, because of its ability to recognize helical distortions, the MMR machinery also binds to damage-containing DNA, specifically triggering checkpoint signaling in higher eukaryotes (4).In terms of the contribution of DNA damage to mutagenesis, some lesions are miscoding and promote the insertion of an incorrect nucleotide (5). Other lesions such as abasic sites and bulky covalent attachments, however, completely block the progress of replicative DNA polymerases in vitro. Such polymerase-blocking lesions must be bypassed in vivo to avoid cell cycle arrest and possible apoptosis. One type of bypass involves insertion of a nucleotide opposite the blocking lesion by a specialized translesion synthesis (TLS) DNA polymerase. Such bypass can be error-free or error-prone depending on the specific TLS polymerase recruited and/or the nature of the lesion (6, 7). The second ty...

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Analysis of Damage Tolerance Pathways in Saccharomyces cerevisiae: a Requirement for Rev3 DNA Polymerase in Translesion Synthesis

Cited by 75 publications

References 46 publications

DNA Repair Defects Channel Interstrand DNA Cross-links into Alternate Recombinational and Error-prone Repair Pathways

DNA Repair Defects Channel Interstrand DNA Cross-links into Alternate Recombinational and Error-prone Repair Pathways

Requirement of Rad5 for DNA Polymerase ζ-Dependent Translesion Synthesis in Saccharomyces cerevisiae

The mismatch repair system promotes DNA polymerase ζ-dependent translesion synthesis in yeast

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