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1.  Topoisomerase I Plays a Critical Role in Suppressing Genome Instability at a Highly Transcribed G-Quadruplex-Forming Sequence 
PLoS Genetics  2014;10(12):e1004839.
G-quadruplex or G4 DNA is a non-B secondary DNA structure that comprises a stacked array of guanine-quartets. Cellular processes such as transcription and replication can be hindered by unresolved DNA secondary structures potentially endangering genome maintenance. As G4-forming sequences are highly frequent throughout eukaryotic genomes, it is important to define what factors contribute to a G4 motif becoming a hotspot of genome instability. Using a genetic assay in Saccharomyces cerevisiae, we previously demonstrated that a potential G4-forming sequence derived from a guanine-run containing immunoglobulin switch Mu (Sμ) region becomes highly unstable when actively transcribed. Here we describe assays designed to survey spontaneous genome rearrangements initiated at the Sμ sequence in the context of large genomic areas. We demonstrate that, in the absence of Top1, a G4 DNA-forming sequence becomes a strong hotspot of gross chromosomal rearrangements and loss of heterozygosity associated with mitotic recombination within the ∼20 kb or ∼100 kb regions of yeast chromosome V or III, respectively. Transcription confers a critical strand bias since genome rearrangements at the G4-forming Sμ are elevated only when the guanine-runs are located on the non-transcribed strand. The direction of replication and transcription, when in a head-on orientation, further contribute to the elevated genome instability at a potential G4 DNA-forming sequence. The implications of our identification of Top1 as a critical factor in suppression of instability associated with potential G4 DNA-forming sequences are discussed.
Author Summary
Genome instability is not evenly distributed, but rather is highly elevated at certain genomic loci containing DNA sequences that can fold into non-canonical secondary structures. The four-stranded G-quadruplex or G4 DNA is one such DNA structure capable of instigating transcription and/or replication obstruction and subsequent genome instability. In this study, we used a reporter system to quantitatively measure the level of genome instability occurring at a G4 DNA motif integrated into the yeast genome. We showed that the disruption of Topoisomerase I function significantly elevated various types of genome instability at the highly transcribed G4 motif generating loss of heterozygosity and copy number alterations (deletions and duplications), both of which are frequently observed in cancer genomes.
doi:10.1371/journal.pgen.1004839
PMCID: PMC4256205  PMID: 25473964
2.  Removal of N-6-methyladenine by the nucleotide excision repair pathway triggers the repair of mismatches in yeast gap-repair intermediates 
DNA repair  2013;12(12):10.1016/j.dnarep.2013.09.006.
Gap-repair assays have been an important tool for studying the genetic control of homologous recombination in yeast. Sequence analysis of recombination products derived when a gapped plasmid is diverged relative to the chromosomal repair template additionally has been used to infer structures of strand-exchange intermediates. In the absence of the canonical mismatch repair pathway, mismatches present in these intermediates are expected to persist and segregate at the next round of DNA replication. In a mismatch repair defective (mlh1Δ) background, however, we have observed that recombination-generated mismatches are often corrected to generate gene conversion or restoration events. In the analyses reported here, the source of the aberrant mismatch removal during gap repair was examined. We find that most mismatch removal is linked to the methylation status of the plasmid used in the gap-repair assay. Whereas more than half of Dam-methylated plasmids had patches of gene conversion and/or restoration interspersed with unrepaired mismatches, mismatch removal was observed in less than 10% of products obtained when un-methylated plasmids were used in transformation experiments. The methylation-linked removal of mismatches in recombination intermediates was due specifically to the nucleotide excision repair pathway, with such mismatch removal being partially counteracted by glycosylases of the base excision repair pathway. These data demonstrate that nucleotide excision repair activity is not limited to bulky, helix-distorting DNA lesions, but also targets removal of very modest perturbations in DNA structure. In addition to its effects on mismatch removal, methylation reduced the overall gap-repair efficiency, but this reduction was not affected by the status of excision repair pathways. Finally, gel purification of DNA prior to transformation reduced gap-repair efficiency four-fold in a nucleotide excision repair-defective background, indicating that the cillateral introduction of UV damage can potentially compromise genetic interpretations.
doi:10.1016/j.dnarep.2013.09.006
PMCID: PMC3858172  PMID: 24120148
transformation; mismatch repair; recombination; methylation; nucleotide excision repair; base excision repair
3.  Roles of exonucleases and translesion synthesis DNA polymerases during mitotic gap repair in yeast 
DNA repair  2013;12(12):10.1016/j.dnarep.2013.10.001.
Transformation-based gap-repair assays have long been used to model the repair of mitotic double-strand breaks (DSBs) by homologous recombination in yeast. In the current study, we examine genetic requirements of two key processes involved in DSB repair: (1) the processive 5′-end resection that is required to efficiently engage a repair template and (2) the filling of resected ends by DNA polymerases. The specific gap-repair assay used allows repair events resolved as crossover versus noncrossover products to be distinguished, as well as the extent of heteroduplex DNA formed during recombination to be measured. To examine end resection, the efficiency and outcome of gap repair were monitored in the absence of the Exo1 exonuclease and the Sgs1 helicase. We found that either Exo1 or Sgs1 presence is sufficient to inhibit gap-repair efficiency over 10-fold, consistent with resection-mediated destruction of the introduced plasmid. In terms of DNA polymerase requirements for gap repair, we focused specifically on potential roles of the Pol ζ and Pol η translesion synthesis DNA polymerases. We found that both Pol ζ and Pol η are necessary for efficient gap repair and that each functions independently of the other. These polymerases may be either in the initiation of DNA synthesis from the an invading end, or in a gap-filling process that is required to complete recombination.
doi:10.1016/j.dnarep.2013.10.001
PMCID: PMC3870910  PMID: 24210827
Sgs1; Exo1; Pol ζ; Pol η; recombination
4.  Two distinct mechanisms of Topoisomerase 1-dependent mutagenesis in yeast 
DNA repair  2013;12(3):205-211.
Topoisomerase 1 (Top1) resolves transcription-associated supercoils by generating transient single-strand breaks in DNA. Top1 activity in yeast is a major source of transcription-associated mutagenesis, generating a distinctive mutation signature characterized by deletions in short, tandem repeats. A similar signature is associated with the persistence of ribonucleoside monophosphates (rNMPs) in DNA, and it also depends on Top1 activity. There is only partial overlap, however, between Top1-dependent deletion hotspots identified in highly transcribed DNA and those associated with rNMPs, suggesting the existence of both rNMP-dependent and rNMP-independent events. Here, we present genetic studies confirming that there are two distinct types of hotspots. Data suggest a novel model in which rNMP-dependent hotspots are generated by sequential Top1 reactions and are consistent with rNMP-independent hotspots reflecting processing of a trapped Top1 cleavage complex.
doi:10.1016/j.dnarep.2012.12.004
PMCID: PMC3594648  PMID: 23305949
Transcription-associated mutagenesis; topoisomerase 1; mutagenesis; ribonucleotide
5.  RNA∶DNA Hybrids Initiate Quasi-Palindrome-Associated Mutations in Highly Transcribed Yeast DNA 
PLoS Genetics  2013;9(11):e1003924.
RNase H enzymes promote genetic stability by degrading aberrant RNA∶DNA hybrids and by removing ribonucleotide monophosphates (rNMPs) that are present in duplex DNA. Here, we report that loss of RNase H2 in yeast is associated with mutations that extend identity between the arms of imperfect inverted repeats (quasi-palindromes or QPs), a mutation type generally attributed to a template switch during DNA synthesis. QP events were detected using frameshift-reversion assays and were only observed under conditions of high transcription. In striking contrast to transcription-associated short deletions that also are detected by these assays, QP events do not require Top1 activity. QP mutation rates are strongly affected by the direction of DNA replication and, in contrast to their elevation in the absence of RNase H2, are reduced when RNase H1 is additionally eliminated. Finally, transcription-associated QP events are limited by components of the nucleotide excision repair pathway and are promoted by translesion synthesis DNA polymerases. We suggest that QP mutations reflect either a transcription-associated perturbation of Okazaki-fragment processing, or the use of a nascent transcript to resume replication following a transcription-replication conflict.
Author Summary
Mutation rates are correlated with the level of gene expression in budding yeast, demonstrating a link between transcription and stability of the underlying DNA template. In the current work, we describe a novel type of transcription-associated mutation that converts imperfect inverted repeats (quasi-palindromes or QPs) to perfect inverted repeats. Using appropriate mutation reporters, we demonstrate that QP mutations are strongly affected by the direction of DNA replication and have distinctive genetic requirements. Most notably, rates of transcription-associated QP events are regulated by the RNase H class of enzymes, which are specialized to process the RNA component of RNA∶DNA hybrids. The source of the RNA∶DNA hybrids that initiate QP mutations is unclear, but could reflect transcripts that remain stably base-paired with the DNA template, or aberrant processing of the RNA primers normally used to initiate DNA synthesis. These studies further expand the diverse ways that transcription affects the mutation landscape, and establish a novel way that RNA∶DNA hybrids can contribute to genetic instability. The high conservation of basic DNA-related metabolic processes suggests that results in yeast will be broadly applicable in higher eukaryotes.
doi:10.1371/journal.pgen.1003924
PMCID: PMC3820800  PMID: 24244191
6.  Heteroduplex DNA Position Defines the Roles of the Sgs1, Srs2, and Mph1 Helicases in Promoting Distinct Recombination Outcomes 
PLoS Genetics  2013;9(3):e1003340.
The contributions of the Sgs1, Mph1, and Srs2 DNA helicases during mitotic double-strand break (DSB) repair in yeast were investigated using a gap-repair assay. A diverged chromosomal substrate was used as a repair template for the gapped plasmid, allowing mismatch-containing heteroduplex DNA (hDNA) formed during recombination to be monitored. Overall DSB repair efficiencies and the proportions of crossovers (COs) versus noncrossovers (NCOs) were determined in wild-type and helicase-defective strains, allowing the efficiency of CO and NCO production in each background to be calculated. In addition, the products of individual NCO events were sequenced to determine the location of hDNA. Because hDNA position is expected to differ depending on whether a NCO is produced by synthesis-dependent-strand-annealing (SDSA) or through a Holliday junction (HJ)–containing intermediate, its position allows the underlying molecular mechanism to be inferred. Results demonstrate that each helicase reduces the proportion of CO recombinants, but that each does so in a fundamentally different way. Mph1 does not affect the overall efficiency of gap repair, and its loss alters the CO-NCO by promoting SDSA at the expense of HJ–containing intermediates. By contrast, Sgs1 and Srs2 are each required for efficient gap repair, strongly promoting NCO formation and having little effect on CO efficiency. hDNA analyses suggest that all three helicases promote SDSA, and that Sgs1 and Srs2 additionally dismantle HJ–containing intermediates. The hDNA data are consistent with the proposed role of Sgs1 in the dissolution of double HJs, and we propose that Srs2 dismantles nicked HJs.
Author Summary
Chromosomal damage that occurs during normal cell division can be repaired using an intact sequence elsewhere in the genome as a template. This process, termed homologous recombination, is crucial for the repair of a particularly deleterious lesion, the DNA double-strand break. Although recombination is a repair process, it can also lead to exchanges of genetic material, generating crossovers (COs) between the involved chromosomes. Repair of the break without exchange of flanking DNA is called a noncrossover (NCO). As COs can uncover recessive mutations or result in large-scale genome rearrangements, understanding how the CO-NCO outcome is regulated is critical to issues of genome stability. The current study examines the distinctive mechanisms whereby three yeast DNA helicases—Mph1, Sgs1, and Srs2—contribute to the repair of a DNA double-strand break.
doi:10.1371/journal.pgen.1003340
PMCID: PMC3597516  PMID: 23516370
7.  Formaldehyde-induced mutagenesis in Saccharomyces cerevisiae: molecular properties and the roles of repair and bypass systems 
Mutation research  2011;731(1-2):92-98.
Although DNA-protein cross-links (DPCs) pose a significant threat to genome stability, they remain a poorly understood class of DNA lesions. To define genetic impacts of DPCs on eukaryotic cells in molecular terms, we used a sensitive Saccharomyces cerevisiae frameshift-detection assay to analyze mutagenesis by formaldehyde (HCHO), and its response to nucleotide excision repair (NER) and translesion DNA synthesis (TLS). Brief exposure to HCHO was mutagenic for NER-defective rad14 strains but not for a corresponding RAD14 strain, nor for a rad14 strain lacking both Polζ and Polη TLS polymerases. This confirmed that HCHO-generated DNA lesions can trigger error-prone TLS and are substrates for the NER pathway. Sequencing revealed that HCHO-induced single-base-pair insertions occurred primarily at one hotspot; most of these insertions were also complex, changing an additional base-pair nearby. Most of the HCHO-induced mutations required both Polζ and Polη, providing a striking example of cooperativity between these two TLS polymerases during bypass of a DNA lesion formed in vivo. The similar molecular properties of HCHO-induced and spontaneous complex +1 insertions detected by this system suggest that DPCs which form in vivo during normal metabolism may contribute characteristic events to the spectra of spontaneous mutations in NER-deficient cells.
doi:10.1016/j.mrfmmm.2011.12.004
PMCID: PMC3591480  PMID: 22197481
DNA-protein cross-link; frameshift mutations; Complex insertions; DNA Polymerase ζ; DNA Polymerase η
8.  The dCMP transferase activity of yeast Rev1 is biologically relevant during the bypass of endogenously generated AP sites 
DNA repair  2011;10(12):1262-1271.
The bypass of AP sites in yeast requires the Rev1 protein in addition to the Pol ζ translesion synthesis DNA polymerase. Although Rev1 was originally characterized biochemically as a dCMP transferase during AP-site bypass, the relevance of this activity in vivo is unclear. The current study uses highly sensitive frameshift- and nonsense-reversion assays to monitor the bypass of AP sites created when uracil is excised from chromosomal DNA. In the frameshift-reversion assay, an unselected base substitution frequently accompanies the selected mutation, allowing the relative incorporation of each of the four dNMPs opposite endogenously created AP sites to be inferred. Results with this assay suggest that dCMP is the most frequent dNMP inserted opposite uracil-derived AP sites and demonstrate that dCMP insertion absolutely requires the catalytic activity of Rev1. In the complementary nonsense-reversion assay, dCMP insertion likewise depended on the dCMP transferase activity of Rev1. Because dAMP insertion opposite uracil-derived AP sites does not revert the nonsense allele and hence could not be detected, it also was possible to detect low levels of dGMP or dTMP insertion upon loss of Rev1 catalytic activity. These results demonstrate that the catalytic activity of Rev1 is biologically relevant and is required specifically for dCMP insertion during the bypass of endogenous AP sites.
doi:10.1016/j.dnarep.2011.09.017
PMCID: PMC3229159  PMID: 22024240
Polζ; Rev1; AP sites; mutagenesis; translesion synthesis
9.  Guanine repeat-containing sequences confer transcription-dependent instability in an orientation-specific manner in yeast 
DNA repair  2011;10(9):953-960.
Non-B DNA structures are a major contributor to the genomic instability associated with repetitive sequences. Immunoglobulin switch Mu (Sμ) region sequence is comprised of guanine-rich repeats and has high potential for forming G4 DNA, in which one strand of DNA folds into an array of guanine quartets. Taking advantage of the genetic tractability of Saccharomyces cerevisiae, we developed a recombination assay to investigate mechanisms involved in maintaining stability of G-rich repetitive sequence. By embedding Sμ sequence within recombination substrates under the control of a tetracycline-regulatable promoter, we demonstrate that the rate and orientation of transcription both affect the stability of Sμ sequence. In particular, the greatest instability was observed under high-transcription conditions when the Sμ sequence was oriented with the C-rich strand as the transcription template. The effect of transcription orientation was enhanced in the absence of the Type IB topoiosmerase Top1, possibly due to enhanced R-loop formation. Loss of Sgs1 helicase and RNase H activity also increased instability, suggesting they may cooperatively function to reduce the formation of non-B DNA structures in highly transcribed regions. Finally, the Sμ sequence was unstable when transcription elongation was perturbed due to a defective THO complex. In a THO-deficient background, there was further exacerbation of orientation-dependent instability associated with the ectopically expressed, single-strand cytosine deaminase AID. The implications of our findings to understanding instability associated with potential G4 DNA forming sequences are discussed.
doi:10.1016/j.dnarep.2011.07.002
PMCID: PMC3162091  PMID: 21813340
G4 DNA; R-loops; genome instability; transcription; recombination
10.  Mutagenic processing of ribonucleotides in DNA by yeast topoisomerase 1 
Science (New York, N.y.)  2011;332(6037):1561-1564.
The RNase H class of enzymes degrades the RNA component of RNA:DNA hybrids and is important in nucleic acid metabolism. RNase H2 is specialized to remove single ribonucleotides (rNMPs) from duplex DNA, and its absence in budding yeast has been associated with the accumulation of deletions within short tandem repeats. Here, we demonstrate that rNMP-associated deletion formation requires the activity of Top1, a topoisomerase that relaxes supercoils by reversibly nicking duplex DNA. The reported studies extend the role of Top1 to include the processing of rNMPs in genomic DNA into irreversible single-strand breaks, an activity that can have distinct mutagenic consequences and may be relevant to human disease.
doi:10.1126/science.1205016
PMCID: PMC3380281  PMID: 21700875
11.  Transcription as a source of genome instability 
Nature Reviews. Genetics  2012;13(3):204-214.
Alterations in genome sequence and structure contribute to somatic disease, affect the fitness of subsequent generations and drive evolutionary processes. The critical roles of highly accurate replication and efficient repair in maintaining overall genome integrity are well known, but the more localized stability costs associated with transcribing DNA into RNA molecules are less appreciated. Here we review the diverse ways that the essential process of transcription alters the underlying DNA template and thereby modifies the genetic landscape.
doi:10.1038/nrg3152
PMCID: PMC3376450  PMID: 22330764
12.  Molecular structures of crossover and noncrossover intermediates during gap repair in yeast: implications for recombination mechanisms and the regulation of recombination fidelity 
Molecular cell  2010;38(2):211-222.
SUMMARY
The molecular structures of crossover (CO) and noncrossover (NCO) intermediates were determined by sequencing the products formed when a gapped plasmid was repaired using a diverged chromosomal template. Analyses were done in the absence of mismatch repair (MMR) to allow efficient detection of strand-transfer intermediates, and the results reveal striking differences in the extents and locations of heteroduplex DNA (hDNA) in NCO versus CO products. These data indicate that most NCOs are produced by synthesis-dependent strand annealing rather than by a canonical double-strand break repair pathway, and that resolution of Holliday junctions formed as part of the latter pathway is highly constrained to generate CO products. We suggest a model in which the length of hDNA formed by the initiating strand invasion event determines susceptibility of the resulting intermediate to antirecombination and ultimately whether a CO- or a NCO-producing pathway is followed.
doi:10.1016/j.molcel.2010.02.028
PMCID: PMC2865147  PMID: 20417600
13.  Abasic Sites in the Transcribed Strand of Yeast DNA Are Removed by Transcription-Coupled Nucleotide Excision Repair▿ † 
Molecular and Cellular Biology  2010;30(13):3206-3215.
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.
doi:10.1128/MCB.00308-10
PMCID: PMC2897580  PMID: 20421413
14.  The Polymerase η Translesion Synthesis DNA Polymerase Acts Independently of the Mismatch Repair System To Limit Mutagenesis Caused by 7,8-Dihydro-8-Oxoguanine in Yeast▿ †  
Molecular and Cellular Biology  2009;29(19):5316-5326.
Reactive oxygen species are ubiquitous mutagens that have been linked to both disease and aging. The most studied oxidative lesion is 7,8-dihydro-8-oxoguanine (GO), which is often miscoded during DNA replication, resulting specifically in GC → TA transversions. In yeast, the mismatch repair (MMR) system repairs GO·A mismatches generated during DNA replication, and the polymerase η (Polη) translesion synthesis DNA polymerase additionally promotes error-free bypass of GO lesions. It has been suggested that Polη limits GO-associated mutagenesis exclusively through its participation in the filling of MMR-generated gaps that contain GO lesions. In the experiments reported here, the SUP4-o forward-mutation assay was used to monitor GC → TA mutation rates in strains defective in MMR (Msh2 or Msh6) and/or in Polη activity. The results clearly demonstrate that Polη can function independently of the MMR system to prevent GO-associated mutations, presumably through preferential insertion of cytosine opposite replication-blocking GO lesions. Furthermore, the Polη-dependent bypass of GO lesions is more efficient on the lagging strand of replication and requires an interaction with proliferating cell nuclear antigen. These studies establish a new paradigm for the prevention of GO-associated mutagenesis in eukaryotes.
doi:10.1128/MCB.00422-09
PMCID: PMC2747974  PMID: 19635811
15.  dUTP incorporation into genomic DNA is linked to transcription in yeast 
Nature  2009;459(7250):1150-1153.
Highly-activated transcription is associated with eukaryotic genome instability, resulting in elevated rates of mitotic recombination and mutagenesis. The association between high transcription and genome stability is likely due to a variety of factors including an enhanced accumulation of DNA damage, transcription-associated supercoiling, collision between replication forks and the transcription machinery, and the persistence of RNA-DNA hybrids 1. In the case of transcription-associated mutagenesis (TAM), we previously showed that there is a direct proportionality between the level of transcription and the mutation rate in the yeast Saccharomyces cerevisiae2, and that the molecular nature of mutations is affected by highly-activated transcription 2 3. In the work presented here, we find that the accumulation of apurinic/apyrimidinic (AP) sites is greatly enhanced in highly-transcribed yeast DNA. We further demonstrate that most AP sites in highly-transcribed DNA are derived from the removal of uracil, the presence of which is linked to direct incorporation of dUTP in place of dTTP. These results reveal an unexpected relationship between transcription and the fidelity of DNA synthesis, and raise intriguing cell biological issues with regard to nucleotide pool compartmentalization.
doi:10.1038/nature08033
PMCID: PMC2730915  PMID: 19448611
16.  TRANSCRIPTION-ASSOCIATED MUTAGENESIS IN YEAST IS DIRECTLY PROPORTIONAL TO THE LEVEL OF GENE EXPRESSION AND INFLUENCED BY THE DIRECTION OF DNA REPLICATION 
DNA repair  2007;6(9):1285-1296.
A high level of transcription has been associated with elevated spontaneous mutation and recombination rates in eukaryotic organisms. To determine whether the transcription level is directly correlated with the degree of genomic instability, we have developed a tetracycline-regulated LYS2 reporter system to modulate the transcription level over a broad range in Saccharomyces cerevisiae. We find that spontaneous mutation rate is directly proportional to the transcription level, suggesting that movement of RNA polymerase through the target initiates a mutagenic process(es). Using this system, we also investigated two hypotheses that have been proposed to explain transcription-associated mutagenesis (TAM): 1) transcription impairs replication fork progression in a directional manner and 2) DNA lesions accumulate under high-transcription conditions. The effect of replication fork progression was probed by comparing the mutational rates and spectra in yeast strains with the reporter gene placed in two different orientations near a well-characterized replication origin. The effect of endogenous DNA damage accumulation was investigated by studying TAM in strains defective in nucleotide excision repair or in lesion bypass by the translesion polymerase Polζ. Our results suggest that both replication orientation and endogenous lesion accumulation play significant roles in TAM, particularly in terms of mutation spectra.
doi:10.1016/j.dnarep.2007.02.023
PMCID: PMC2034516  PMID: 17398168
spontaneous mutagenesis; transcription; replication direction; polymerase zeta; mutation spectrum
17.  The effect of sequence context on spontaneous Polζ-dependent mutagenesis in Saccharomyces cerevisiae 
Nucleic Acids Research  2008;36(6):2082-2093.
The Polζ translesion synthesis (TLS) DNA polymerase is responsible for over 50% of spontaneous mutagenesis and virtually all damage-induced mutagenesis in yeast. We previously demonstrated that reversion of the lys2ΔA746 −1 frameshift allele detects a novel type of +1 frameshift that is accompanied by one or more base substitutions and depends completely on the activity of Polζ. These ‘complex’ frameshifts accumulate at two discrete hotspots (HS1 and HS2) in the absence of nucleotide excision repair, and accumulate at a third location (HS3) in the additional absence of the translesion polymerase Polη. The current study investigates the sequence requirements for accumulation of Polζ-dependent complex frameshifts at these hotspots. We observed that transposing 13 bp of identity from HS1 or HS3 to a new location within LYS2 was sufficient to recapitulate these hotspots. In addition, altering the sequence immediately upstream of HS2 had no effect on the activity of the hotspot. These data support a model in which misincorporation opposite a lesion precedes and facilitates the selected slippage event. Finally, analysis of nonsense mutation revertants indicates that Polζ can simultaneously introduce multiple base substitutions in the absence of an accompanying frameshift event.
doi:10.1093/nar/gkn054
PMCID: PMC2330250  PMID: 18276637
18.  Novel PMS1 Alleles Preferentially Affect the Repair of Primer Strand Loops during DNA Replication 
Molecular and Cellular Biology  2005;25(21):9221-9231.
Null mutations in DNA mismatch repair (MMR) genes elevate both base substitutions and insertions/deletions in simple sequence repeats. Data suggest that during replication of simple repeat sequences, polymerase slippage can generate single-strand loops on either the primer or template strand that are subsequently processed by the MMR machinery to prevent insertions and deletions, respectively. In the budding yeast Saccharomyces cerevisiae and mammalian cells, MMR appears to be more efficient at repairing mispairs comprised of loops on the template strand compared to loops on the primer strand. We identified two novel yeast pms1 alleles, pms1-G882E and pms1-H888R, which confer a strong defect in the repair of “primer strand” loops, while maintaining efficient repair of “template strand” loops. Furthermore, these alleles appear to affect equally the repair of 1-nucleotide primer strand loops during both leading- and lagging-strand replication. Interestingly, both pms1 mutants are proficient in the repair of 1-nucleotide loop mispairs in heteroduplex DNA generated during meiotic recombination. Our results suggest that the inherent inefficiency of primer strand loop repair is not simply a mismatch recognition problem but also involves Pms1 and other proteins that are presumed to function downstream of mismatch recognition, such as Mlh1. In addition, the findings reinforce the current view that during mutation avoidance, MMR is associated with the replication apparatus.
doi:10.1128/MCB.25.21.9221-9231.2005
PMCID: PMC1265805  PMID: 16227575
19.  Mutagenic effects of abasic and oxidized abasic lesions in Saccharomyces cerevisiae 
Nucleic Acids Research  2005;33(19):6196-6202.
2-Deoxyribonolactone (L) and 2-deoxyribose (AP) are abasic sites that are produced by ionizing radiation, reactive oxygen species and a variety of DNA damaging agents. The biological processing of the AP site has been examined in the yeast Saccharomyces cerevisiae. However, nothing is known about how L is processed in this organism. We determined the bypass and mutagenic specificity of DNA containing an abasic site (AP and L) or the AP analog tetrahydrofuran (F) using an oligonucleotide transformation assay. The tetrahydrofuran analog and L were bypassed at 10-fold higher frequencies than the AP lesions. Bypass frequencies of lesions were greatly reduced in the absence of Rev1 or Polζ (rev3 mutant), but were only marginally reduced in the absence of Polη (rad30 mutant). Deoxycytidine was the preferred nucleotide inserted opposite an AP site whereas dA and dC were inserted at equal frequencies opposite F and L sites. In the rev1 and rev3 strains, dA was the predominant nucleotide inserted opposite these lesions. Overall, we conclude that both Rev1 and Polζ are required for the efficient bypass of abasic sites in yeast.
doi:10.1093/nar/gki926
PMCID: PMC1275587  PMID: 16257982
20.  Identification of a Distinctive Mutation Spectrum Associated with High Levels of Transcription in Yeast 
Molecular and Cellular Biology  2004;24(11):4801-4809.
High levels of transcription are associated with increased mutation rates in Saccharomyces cerevisiae, a phenomenon termed transcription-associated mutation (TAM). To obtain insight into the mechanism of TAM, we obtained LYS2 forward mutation spectra under low- versus high-transcription conditions in which LYS2 was expressed from either the low-level pLYS2 promoter or the strong pGAL1-10 promoter, respectively. Because of the large size of the LYS2 locus, forward mutations first were mapped to specific LYS2 subregions, and then those mutations that occurred within a defined 736-bp target region were sequenced. In the low-transcription strain base substitutions comprised the majority (64%) of mutations, whereas short insertion-deletion mutations predominated (56%) in the high-transcription strain. Most notably, deletions of 2 nucleotides (nt) comprised 21% of the mutations in the high-transcription strain, and these events occurred predominantly at 5′-(G/C)AAA-3′ sites. No −2 events were present in the low-transcription spectrum, thus identifying 2-nt deletions as a unique mutational signature for TAM.
doi:10.1128/MCB.24.11.4801-4809.2004
PMCID: PMC416428  PMID: 15143174
21.  Base Composition of Mononucleotide Runs Affects DNA Polymerase Slippage and Removal of Frameshift Intermediates by Mismatch Repair in Saccharomyces cerevisiae 
Molecular and Cellular Biology  2002;22(24):8756-8762.
The postreplicative mismatch repair (MMR) system is important for removing mutational intermediates that are generated during DNA replication, especially those that arise as a result of DNA polymerase slippage in simple repeats. Here, we use a forward mutation assay to systematically examine the accumulation of frameshift mutations within mononucleotide runs of variable composition in wild-type and MMR-defective yeast strains. These studies demonstrate that (i) DNA polymerase slippage occurs more often in 10-cytosine/10-guanine (10C/10G) runs than in 10-adenine/10-thymine (10A/10T) runs, (ii) the MMR system removes frameshift intermediates in 10A/10T runs more efficiently than in 10C/10G runs, (iii) the MMR system removes −1 frameshift intermediates more efficiently than +1 intermediates in all 10-nucleotide runs, and (iv) the repair specificities of the Msh2p-Msh3p and Msh2p-Msh6p mismatch recognition complexes with respect to 1-nucleotide insertion/deletion loops vary dramatically as a function of run composition. These observations are relevant to issues of genome stability, with both the rates and types of mutations that accumulate in mononucleotide runs being influenced by the primary sequence of the run as well as by the status of the MMR system.
doi:10.1128/MCB.22.24.8756-8762.2002
PMCID: PMC139878  PMID: 12446792
22.  Stimulation of Mitotic Recombination Events by High Levels of RNA Polymerase II Transcription in Yeast 
Molecular and Cellular Biology  2000;20(15):5404-5414.
The impact of high levels of RNA polymerase II transcription on mitotic recombination was examined using lys2 recombination substrates positioned on nonhomologous chromosomes. Substrates were used that could produce Lys+ recombinants by either a simple (noncrossover) gene conversion event or a crossover-associated recombination event, by only a simple gene conversion event, or by only a crossover event. Transcription of the lys2 substrates was regulated by the highly inducible GAL1-10 promoter or the low-level LYS2 promoter, with GAL1-10 promoter activity being controlled by the presence or absence of the Gal80p negative regulatory protein. Transcription was found to stimulate recombination in all assays used, but the level of stimulation varied depending on whether only one or both substrates were highly transcribed. In addition, there was an asymmetry in the types of recombination events observed when one substrate versus the other was highly transcribed. Finally, the lys2 substrates were positioned as direct repeats on the same chromosome and were found to exhibit a different recombinational response to high levels of transcription from that exhibited by the repeats on nonhomologous chromosomes. The relevance of these results to the mechanisms of transcription-associated recombination are discussed.
PMCID: PMC85992  PMID: 10891481
23.  Removal of Frameshift Intermediates by Mismatch Repair Proteins in Saccharomyces cerevisiae 
Molecular and Cellular Biology  1999;19(7):4766-4773.
Frameshift mutations occur when the coding region of a gene is altered by addition or deletion of a number of base pairs that is not a multiple of three. The occurrence of a deletion versus an insertion type of frameshift depends on the nature of the transient intermediate structure formed during DNA synthesis. Extrahelical bases on the template strand give rise to deletions, whereas extrahelical bases on the strand being synthesized produce insertions. We previously used reversion of a +1 frameshift mutation to analyze the role of the mismatch repair (MMR) machinery in correcting −1 frameshift intermediates within a defined region of the yeast LYS2 gene. In this study, we have used reversion of a −1 frameshift mutation within the same region of LYS2 to analyze the role of the MMR machinery in the correction of frameshift intermediates that give rise to insertion events. We found that insertion and deletion events occur at similar rates but that the reversion spectra are very different in both the wild-type and MMR-defective backgrounds. In addition, analysis of the +1 spectra revealed novel roles for Msh3p and Msh6p in removing specific types of frameshift intermediates.
PMCID: PMC84275  PMID: 10373526
24.  Overlapping Specificities of Base Excision Repair, Nucleotide Excision Repair, Recombination, and Translesion Synthesis Pathways for DNA Base Damage in Saccharomyces cerevisiae 
Molecular and Cellular Biology  1999;19(4):2929-2935.
The removal of oxidative damage from Saccharomyces cerevisiae DNA is thought to be conducted primarily through the base excision repair pathway. The Escherichia coli endonuclease III homologs Ntg1p and Ntg2p are S. cerevisiae N-glycosylase-associated apurinic/apyrimidinic (AP) lyases that recognize a wide variety of damaged pyrimidines (H. J. You, R. L. Swanson, and P. W. Doetsch, Biochemistry 37:6033–6040, 1998). The biological relevance of the N-glycosylase-associated AP lyase activity in the repair of abasic sites is not well understood, and the majority of AP sites in vivo are thought to be processed by Apn1p, the major AP endonuclease in yeast. We have found that yeast cells simultaneously lacking Ntg1p, Ntg2p, and Apn1p are hyperrecombinogenic (hyper-rec) and exhibit a mutator phenotype but are not sensitive to the oxidizing agents H2O2 and menadione. The additional disruption of the RAD52 gene in the ntg1 ntg2 apn1 triple mutant confers a high degree of sensitivity to these agents. The hyper-rec and mutator phenotypes of the ntg1 ntg2 apn1 triple mutant are further enhanced by the elimination of the nucleotide excision repair pathway. In addition, removal of either the lesion bypass (Rev3p-dependent) or recombination (Rad52p-dependent) pathway specifically enhances the hyper-rec or mutator phenotype, respectively. These data suggest that multiple pathways with overlapping specificities are involved in the removal of, or tolerance to, spontaneous DNA damage in S. cerevisiae. In addition, the fact that these responses to induced and spontaneous damage depend upon the simultaneous loss of Ntg1p, Ntg2p, and Apn1p suggests a physiological role for the AP lyase activity of Ntg1p and Ntg2p in vivo.
PMCID: PMC84087  PMID: 10082560
25.  Mismatch Repair Proteins Regulate Heteroduplex Formation during Mitotic Recombination in Yeast 
Molecular and Cellular Biology  1998;18(11):6525-6537.
Mismatch repair (MMR) proteins actively inhibit recombination between diverged sequences in both prokaryotes and eukaryotes. Although the molecular basis of the antirecombination activity exerted by MMR proteins is unclear, it presumably involves the recognition of mismatches present in heteroduplex recombination intermediates. This recognition could be exerted during the initial stage of strand exchange, during the extension of heteroduplex DNA, or during the resolution of recombination intermediates. We previously used an assay system based on 350-bp inverted-repeat substrates to demonstrate that MMR proteins strongly inhibit mitotic recombination between diverged sequences in Saccharomyces cerevisiae. The assay system detects only those events that reverse the orientation of the region between the recombination substrates, which can occur as a result of either intrachromatid crossover or sister chromatid conversion. In the present study we sequenced the products of mitotic recombination between 94%-identical substrates in order to map gene conversion tracts in wild-type versus MMR-defective yeast strains. The sequence data indicate that (i) most recombination occurs via sister chromatid conversion and (ii) gene conversion tracts in an MMR-defective strain are significantly longer than those in an isogenic wild-type strain. The shortening of conversion tracts observed in a wild-type strain relative to an MMR-defective strain suggests that at least part of the antirecombination activity of MMR proteins derives from the blockage of heteroduplex extension in the presence of mismatches.
PMCID: PMC109238  PMID: 9774668

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