Mutagenic effects of abasic and oxidized abasic lesions in Saccharomyces cerevisiae

Kow, Yoke W.; Bao, Gaobin; Minesinger, Brenda K.; Jinks-Robertson, Sue; Siede, Wolfram; Jiang, Yu; Greenberg, Marc M.

doi:10.1093/nar/gki926

Cited by 42 publications

(55 citation statements)

References 50 publications

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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: 51%

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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Section: Discussionmentioning

confidence: 51%

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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“…Specifically, the use of ssOligos with differing sensitivities to MutS␣ and MutS␤ has revealed striking and unexpected strand differences in the activity of the two MMR complexes as well as unexpected differences in response to loss of MLH1. With a better understanding of the mechanism in place, this method should prove extremely valuable in studying the effects of defined base mismatches and damage on repair and replication in a chromosomal context (44)(45)(46)(47).…”

Section: Discussionmentioning

confidence: 99%

Oligonucleotide transformation of yeast reveals mismatch repair complexes to be differentially active on DNA replication strands

Kow¹,

Bao²,

Reeves

et al. 2007

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

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Transformation of both prokaryotes and eukaryotes with singlestranded oligonucleotides can transfer sequence information from the oligonucleotide to the chromosome. We have studied this process using oligonucleotides that correct a ؊1 frameshift mutation in the LYS2 gene of Saccharomyces cerevisiae. We demonstrate that transformation by oligonucleotides occurs preferentially on the lagging strand of replication and is strongly inhibited by the mismatch-repair system. These results are consistent with a mechanism in which oligonucleotides anneal to single-stranded regions of DNA at a replication fork and serve as primers for DNA synthesis. Because the mispairs the primers create are efficiently removed by the mismatch-repair system, single-stranded oligonucleotides can be used to probe mismatch-repair function in a chromosomal context. Removal of mispairs created by annealing of the single-stranded oligonucleotides to the chromosomal DNA is as expected, with 7-nt loops being recognized solely by MutS␤ and 1-nt loops being recognized by both MutS␣ and MutS␤. We also find evidence for Mlh1-independent repair of 7-nt, but not 1-nt, loops. Unexpectedly, we find a strand asymmetry of mismatchrepair function; transformation is blocked more efficiently by MutS␣ on the lagging strand of replication, whereas MutS␤ does not show a significant strand bias. These results suggest an inherent strand-related difference in how the yeast MutS␣ and MutS␤ complexes access and/or repair mismatches that arise in the context of DNA replication.T he transformation of yeast by single-stranded oligonucleotides (ssOligos) was first demonstrated 20 years ago by Sherman and colleagues (1). By using the CYC1 gene as a target of ssOligos of 50 nt, it was found that mismatches, in general, reduced ssOligo transformation (ssOT) efficiency, that 3Ј deoxyoligonucleotides transformed somewhat less well than those with a 3Ј hydroxyl and that ssOT was independent of tested recombination functions (2, 3). A strong strand bias also was observed, with ssOligos having the sequence of the coding strand of CYC1 (defined here as COD oligonucleotides) transforming 50-100 times better than oligonucleotides with the complementary sequence (noncoding, or NC, oligonucleotides). It was suggested that the differences between COD and NC ssOT frequencies were not related to transcriptional differences but could be due to preferential incorporation of oligonucleotides into either the leading or lagging strand of replication (3). More recently, the Kmiec (4) lab has studied the transformation of cyc1 point mutants using 70-nt ssOligos with three phosphorothioate bonds at the 3Ј and 5Ј termini. In this case, there appeared to be an opposite strand bias, with NC ssOligos transforming better than COD ssOligos. Furthermore, it was suggested that this transformation was enhanced by repair processes such as mismatch repair (MMR), with transformation apparently decreasing in MMR-defective cells (4).In addition to the yeast studies, it has also been shown that ssOligos can t...

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“…Blocked DNA synthesis can be rescued either by a recombination/template switch mechanism or through the recruitment of specialized translesion synthesis (TLS) DNA polymerases (14). In yeast, TLS polymerase (Pol), together with the deoxycytidyl transferase Rev1, is required for most AP-site bypass, with a C nucleotide usually being inserted across from the template lesion (20,31,39). Depending on the base lost, AP-site bypass via TLS has the potential to be highly mutagenic.…”

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confidence: 99%

Abasic Sites in the Transcribed Strand of Yeast DNA Are Removed by Transcription-Coupled Nucleotide Excision Repair

Kim

Jinks-Robertson

2010

Molecular and Cellular Biology

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Abasic (AP) sites are potent blocks to DNA and RNA polymerases, and their repair is essential for maintaining genome integrity. Although AP sites are efficiently dealt with through the base excision repair (BER) pathway, genetic studies suggest that repair also can occur via nucleotide excision repair (NER). The involvement of NER in AP-site removal has been puzzling, however, as this pathway is thought to target only bulky lesions. Here, we examine the repair of AP sites generated when uracil is removed from a highly transcribed gene in yeast. Because uracil is incorporated instead of thymine under these conditions, the position of the resulting AP site is known. Results demonstrate that only AP sites on the transcribed strand are efficient substrates for NER, suggesting the recruitment of the NER machinery by an AP-blocked RNA polymerase. Such transcription-coupled NER of AP sites may explain previously suggested links between the BER pathway and transcription.High levels of transcription affect genome stability by stimulating mutagenesis and homologous recombination, both of which are well-known consequences of DNA damage (for reviews, see references 2 and 3). Consistently with a link between transcription and DNA damage accumulation, highly transcribed sequences in the yeast Saccharomyces cerevisiae are more susceptible to exogenous mutagens (19), and spontaneous transcription-associated mutagenesis (TAM) is further elevated when DNA repair is compromised (11,30,38). While there likely are multiple causes for and types of damage that accumulate in transcriptionally active DNA, we recently demonstrated that apurinic/apyrimidinic (AP) sites can be a significant source of TAM (30). AP sites are one of the most common DNA lesions and can be a potent block to replicative DNA polymerases (reviewed in reference 15). Blocked DNA synthesis can be rescued either by a recombination/template switch mechanism or through the recruitment of specialized translesion synthesis (TLS) DNA polymerases (14). In yeast, TLS polymerase (Pol), together with the deoxycytidyl transferase Rev1, is required for most AP-site bypass, with a C nucleotide usually being inserted across from the template lesion (20,31,39). Depending on the base lost, AP-site bypass via TLS has the potential to be highly mutagenic.AP sites can be generated by the spontaneous hydrolysis of the sugar base glycosidic linkage or are formed when a specialized DNA N-glycosylase releases its cognate damaged base from the sugar phosphate backbone (reviewed in references 6 and 15). In yeast, genetic studies have demonstrated that most spontaneous AP sites originate from the incorporation of dUTP in place of dTTP during DNA synthesis (22). Subsequent uracil removal by Ung1, the sole uracil DNA glycosylase in S. cerevisiae, creates a potentially toxic/mutagenic AP site. As a part of the cellular defense mechanism against uracil incorporation into DNA, dUTPase, an essential enzyme in yeast (16) as well as in Escherichia coli (13), catalyzes the conversion of dUTP to dUMP. ...

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Mutagenic effects of abasic and oxidized abasic lesions in Saccharomyces cerevisiae

Cited by 42 publications

References 50 publications

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

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

Oligonucleotide transformation of yeast reveals mismatch repair complexes to be differentially active on DNA replication strands

Abasic Sites in the Transcribed Strand of Yeast DNA Are Removed by Transcription-Coupled Nucleotide Excision Repair

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