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

Abstract

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...

DNA Repair Mechanisms and the Bypass of DNA Damage in Saccharomyces cerevisiae

“…Maximal expression of Rev1 outside of S phase has led to the suggestion that most Pol z-dependent lesion bypass occurs via gap-filling reactions that occur well behind the replication fork. Consistent with this, spontaneous, Pol z-dependent lesion bypass is refractory to correction by the MMR machinery (Lehner and Jinks-Robertson 2009). The higher mutation rate of late-replicating DNA also is reduced in rev1D background (Lang and Murray 2011), consistent with the cell-cycle regulation of Rev1 and suggesting that there may be temporal separation of error-free and error-prone lesion bypass.…”

Frameshift Mutagenesis: The Roles of Primer–Template Misalignment and the Nonhomologous End-Joining Pathway inSaccharomyces cerevisiae

“…With regard to the former possibility, we previously reported that suppression of recombination by the MMR system promotes Polz-dependent mutagenesis via the alternative translesion synthesis pathway, making such mutations dependent on functional MMR (Lehner and Jinks-Robertson 2009). We thus examined whether small duplications depend on the presence of Polz.…”

Role for topoisomerase 1 in transcription-associated mutagenesis in yeast

“…lys2ΔA746NR, lys2ΔA746NR,(AT) 2, or lys2ΔA746NR,(TC) 3 derivatives were constructed by two-step allele replacement by using AflIIdigested pSR963 (17), pSR1002, or pSR1003, respectively. pSR1002 and pSR1003 were derived by annealing oligonucleotides [5′-gatcGGTTTTGC-CACATATCTTCAACGCTG and 5′-gatcCAGCGTTGAAGATATGTGGCAAAACC for the (AT) 2 hotspot or 5′-gatcATACCGTGGCATCTCTCGTGACGAGTTAC and 5′-gatcGTAACTCGTCACGAGAGATGCCACGGTAT for the (TC) 3 hotspot; hotspots are underlined, and BglII-compatible overhangs are in lowercase] and then inserting the resulting ≈30 bp fragments into BglII-digested pSR963.…”