Conditions of chronic stress are associated with genetic instability in many organisms, but the roles of stress responses in mutagenesis have so far been elucidated only in bacteria. Here, we present data demonstrating that the environmental stress response (ESR) in yeast functions in mutagenesis induced by proteotoxic stress. We show that the drug canavanine causes proteotoxic stress, activates the ESR, and induces mutagenesis at several loci in an ESR-dependent manner. Canavanine-induced mutagenesis also involves translesion DNA polymerases Rev1 and Polζ and non-homologous end joining factor Ku. Furthermore, under conditions of chronic sub-lethal canavanine stress, deletions of Rev1, Polζ, and Ku-encoding genes exhibit genetic interactions with ESR mutants indicative of ESR regulating these mutagenic DNA repair processes. Analyses of mutagenesis induced by several different stresses showed that the ESR specifically modulates mutagenesis induced by proteotoxic stress. Together, these results document the first known example of an involvement of a eukaryotic stress response pathway in mutagenesis and have important implications for mechanisms of evolution, carcinogenesis, and emergence of drug-resistant pathogens and chemotherapy-resistant tumors.
Cellular capability to mutate its DNA plays an important role in evolution and impinges on medical issues, including acquisition of mutator phenotypes by cancer cells and emergence of drug-resistant pathogens. Whether and how the environment affects rates of mutation has been studied predominantly in the context of environmental agents that damage DNA (e.g. UV and γ-rays). However, it has been observed that conditions of chronic non-DNA-damaging stress (e.g. starvation or heat shock) also increase mutagenesis. It has been shown that in bacteria, activation of the general stress response activates a pro-mutagenic pathway and thus promotes mutagenesis during periods of stress. However, in eukaryotes, so far there has been no evidence of a stress response regulating mutagenesis. In this manuscript we demonstrate that in budding yeast, a model eukaryote, the general environmental stress response (ESR) regulates mutagenesis induced by proteotoxic stress (accumulation of unfolded proteins) at several loci. We also identify two pro-mutagenic DNA metabolic pathways that contribute to this mutagenesis and present genetic data showing that the ESR regulates these pathways. Together, these data advance our understanding of how cellular sensing and responding to environmental cues affect cellular capability for mutagenesis.
8-oxoG is one of the most common and mutagenic DNA base lesions caused by oxidative damage. However, it has not been possible to study the replication of a known 8-oxoG base in vivo in order to determine the accuracy of its replication, the influence of various components on that accuracy, and the extent to which an 8-oxoG might present a barrier to replication. We have been able to place a single 8-oxoG into the Saccharomyces cerevisiae chromosome in a defined location using single-strand oligonucleotide transformation and to study its replication in a fully normal chromosome context. During replication, 8-oxoG is recognized as a lesion and triggers a switch to translesion synthesis by Pol η, which replicates 8-oxoG with an accuracy (insertion of a C opposite the 8-oxoG) of approximately 94%. In the absence of Pol η, template switching to the newly synthesized sister chromatid is observed at least one third of the time; replication of the 8-oxoG in the absence of Pol η is less than 40% accurate. The mismatch repair (MMR) system plays an important role in 8-oxoG replication. Template switching is blocked by MMR and replication accuracy even in the absence of Pol η is approximately 95% when MMR is active. These findings indicate that in light of the overlapping mechanisms by which errors in 8-oxoG replication can be avoided in the cell, the mutagenic threat of 8-oxoG is due more to its abundance than the effect of a single lesion. In addition, the methods used here should be applicable to the study of any lesion that can be stably incorporated into synthetic oligonucleotides.
In the course of normal cellular functions, many types of reactive oxygen species are produced that can lead to oxidative damage in the cell. DNA bases are subject to the formation of various oxidative lesions; one of the most common is the production of 8-oxoG which can pair relatively well with A instead of C, leading to GC→TA transversion mutations. In this work, we have been able to place a single 8-oxoG in the yeast chromosome and observe its replication. We find that in a wild-type cell its replication is surprisingly accurate due primarily to two components: DNA mismatch repair, which recognizes an A inserted opposite the 8-oxoG and initiates its removal and subsequent re-replication; and translesion synthesis in which the existence of the 8-oxoG induces a switch from a normal replicating DNA polymerase to a specialized DNA polymerase, Pol η, that can accurately replicate an 8-oxoG. We also find that the 8-oxoG can cause the replicating strand to shift to the newly replicated strand in the sister chromatid, thus avoiding the 8-oxoG lesion. These findings indicate not only how cells deal with a known DNA lesion, but also demonstrate how other such lesions can be studied in the future.
Non-homologous end-joining (NHEJ) and homologous recombination (HR) represent the two main pathways for repairing DNA double-strand breaks (DSBs). During the G2 phase of the mammalian cell cycle, both processes can operate and chromatin structure is one important factor which determines DSB repair pathway choice. ATM facilitates the repair of heterochromatic DSBs by phosphorylating and inactivating the heterochromatin building factor KAP-1, leading to local chromatin relaxation. Here, we show that ATM accumulation and activity is strongly diminished at DSBs undergoing end-resection during HR. Such DSBs remain unrepaired in cells devoid of the HR factors BRCA2, XRCC3 or RAD51. Strikingly, depletion of KAP-1 or expression of phospho-mimic KAP-1 allows repair of resected DSBs in the absence of BRCA2, XRCC3 or RAD51 by an erroneous PARP-dependent alt-NHEJ process. We suggest that DSBs in heterochromatin elicit initial local heterochromatin relaxation which is reversed during HR due to the release of ATM from resection break ends. The restored heterochromatic structure facilitates HR and prevents usage of error-prone alternative processes.
Double-strand breaks (DSBs) are critical DNA lesions because they can lead to cell death or, which is even more devastating, the formation of genomic rearrangements. Cells are equipped with two main pathways to repair such lesions, homologous recombination (HR) and non-homologous end-joining (NHEJ). HR is an error-free process and completely restores the genetic information, whereas NHEJ has the potential to form genomic rearrangements. We have previously shown that the structure of the chromatin is one important factor which determines the choice between these two pathways, such that DSBs localizing to highly condensed heterochromatic regions are mainly repaired by HR and breaks in more open euchromatic DNA undergo repair by NHEJ. Here, we investigate this aspect of DSB repair pathway choice. We show that DSB end-resection, which channels DSB repair into the process of HR, counteracts the profound local relaxation which initially takes place at the break site and reconstitutes the heterochromatic structure. Cells which are genetically modified, such that they cannot reconstitute the heterochromatic structure at resected DSBs, fail to employ HR and instead repair heterochromatic DSBs by alternative NHEJ mechanisms. Thus, chromatin modifications which occur during the process of end-resection prevent error-prone repair pathways from generating genomic rearrangements.
DNA double-strand breaks (DSBs) are one of the most dangerous DNA lesions, since their erroneous repair by nonhomologous end-joining (NHEJ) can generate harmful chromosomal rearrangements. PolX DNA polymerases are well suited to extend DSB ends that cannot be directly ligated due to their particular ability to bind to and insert nucleotides at the imperfect template-primer structures formed during NHEJ. Herein, we have devised genetic assays in yeast to induce simultaneous DSBs in different chromosomes in vivo. The repair of these breaks in trans could result in reciprocal chromosomal translocations that were dependent on classical Ku-dependent NHEJ. End-joining events leading to translocations were mainly based on the formation of short base pairing between 3′-overhanging DNA ends coupled to gap-filling DNA synthesis. A major proportion of these events were specifically dependent on yeast DNA polymerase Pol4 activity. In addition, we have discovered that Pol4-Thr540 amino acid residue can be phosphorylated by Tel1/ATM kinase, which could modulate Pol4 activity during NHEJ. Our data suggest that the role of Tel1 in preventing break-induced chromosomal translocations can, to some extent, be due to its stimulating effect on gap-filling activity of Pol4 to repair DSBs in cis. Overall, this work provides further insight to the molecular mechanisms of DSB repair by NHEJ and presents a new perspective to the understanding of how chromosomal translocations are formed in eukaryotic cells.
Chromosomal translocations are one of the most common types of genomic rearrangements, which may have a relevant impact on cell development. They are often generated from DNA double-strand breaks that are inaccurately repaired by DNA repair machinery. In this study, we have developed genetic assays in yeast to analyze the molecular mechanisms by which these translocations can arise. We found evidence showing that the classical nonhomologous end-joining repair pathway can be a source of chromosomal translocations, with a relevant role for yeast DNA polymerase Pol4 in such processes. The involvement of Pol4 is based on its efficient gap-filling DNA synthesis activity during the joining of overhanging DNA ends with short sequence complementarity. In addition, we discovered that DNA polymerase Pol4 can be modified during the repair of the breaks via phosphorylation by Tel1 kinase. This phosphorylation seems to have important structural and functional implications in the action of Pol4, which can finally influence the formation of translocations. This work provides a useful tool for deciphering factors and mechanisms involved in DNA double-strand break repair and identifying the molecular pathways leading to chromosomal translocations in eukaryotic cells.
DNA ligase IV (Dnl4 in budding yeast) is a specialized ligase used in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). Although point and truncation mutations arise in the human ligase IV syndrome, the roles of Dnl4 in DSB repair have mainly been examined using gene deletions. Here, Dnl4 catalytic point mutants were generated that were severely defective in auto-adenylation in vitro and NHEJ activity in vivo, despite being hyper-recruited to DSBs and supporting wild-type levels of Lif1 interaction and assembly of a Ku- and Lif1-containing complex at DSBs. Interestingly, residual levels of especially imprecise NHEJ were markedly higher in a deletion-based assay with Dnl4 catalytic mutants than with a gene deletion strain, suggesting a role of DSB-bound Dnl4 in supporting a mode of NHEJ catalyzed by a different ligase. Similarly, next generation sequencing of repair joints in a distinct single-DSB assay showed that dnl4-K466A mutation conferred a significantly different imprecise joining profile than wild-type Dnl4 and that such repair was rarely observed in the absence of Dnl4. Enrichment of DNA ligase I (Cdc9 in yeast) at DSBs was observed in wild-type as well as dnl4 point mutant strains, with both Dnl4 and Cdc9 disappearing from DSBs upon 5′ resection that was unimpeded by the presence of catalytically inactive Dnl4. These findings indicate that Dnl4 can promote mutagenic end joining independently of its catalytic activity, likely by a mechanism that involves Cdc9.
Chromosomal rearrangements are common driver mutations in human genetic disease and cancer. The junctions observed at rearrangements typically show only a few base pairs in common between the partners, suggesting that they were formed by the end-to-end joining process, nonhomologous end joining (NHEJ). However, there is uncertainty about the mechanisms that actually create mutated junctions. DNA ligase IV catalyzes restorative double-strand break (DSB) joining in the canonical NHEJ pathway, but increasing evidence suggests that distinct NHEJ pathways that use DNA ligases I and/or III might be more important for mutations. We used yeast to study the in vivo consequence of having DNA ligase IV that was catalytically inactive but that nonetheless accumulated at DSBs normally. We detected mutated junctions in some assays that required DNA ligase IV protein but not its catalytic activity. This pattern suggests that DNA ligase I creates many mutated junctions when DNA ligase IV is present and that this can become a predominant mode of repair when DNA ligase IV activity is inefficient. Our yeast ligase IV mutations have properties similar to those observed in the human ligase IV syndrome, underscoring the relevance of these observations.
The yeast Saccharomyces cerevisiae is able to accumulate ≥17% ethanol (v/v) by fermentation in the absence of cell proliferation. The genetic basis of this unique capacity is unknown. Up to now, all research has focused on tolerance of yeast cell proliferation to high ethanol levels. Comparison of maximal ethanol accumulation capacity and ethanol tolerance of cell proliferation in 68 yeast strains showed a poor correlation, but higher ethanol tolerance of cell proliferation clearly increased the likelihood of superior maximal ethanol accumulation capacity. We have applied pooled-segregant whole-genome sequence analysis to identify the polygenic basis of these two complex traits using segregants from a cross of a haploid derivative of the sake strain CBS1585 and the lab strain BY. From a total of 301 segregants, 22 superior segregants accumulating ≥17% ethanol in small-scale fermentations and 32 superior segregants growing in the presence of 18% ethanol, were separately pooled and sequenced. Plotting SNP variant frequency against chromosomal position revealed eleven and eight Quantitative Trait Loci (QTLs) for the two traits, respectively, and showed that the genetic basis of the two traits is partially different. Fine-mapping and Reciprocal Hemizygosity Analysis identified ADE1, URA3, and KIN3, encoding a protein kinase involved in DNA damage repair, as specific causative genes for maximal ethanol accumulation capacity. These genes, as well as the previously identified MKT1 gene, were not linked in this genetic background to tolerance of cell proliferation to high ethanol levels. The superior KIN3 allele contained two SNPs, which are absent in all yeast strains sequenced up to now. This work provides the first insight in the genetic basis of maximal ethanol accumulation capacity in yeast and reveals for the first time the importance of DNA damage repair in yeast ethanol tolerance.
The yeast Saccharomyces cerevisiae is unique in being the most ethanol tolerant organism known. This property lies at the basis of its ecological competitiveness in sugar-rich ecological niches and its use for the production of alcoholic beverages and bioethanol, both of which involve accumulation of high levels of ethanol. Up to now, all research on yeast ethanol tolerance has focused on tolerance of cell proliferation to high ethanol levels. However, the most ecologically and industrially relevant aspect is the capacity of fermenting yeast cells to accumulate high ethanol levels in the absence of cell proliferation. Using QTL mapping by pooled-segregant whole-genome sequence analysis, we show that maximal ethanol accumulation capacity and tolerance of cell proliferation to high ethanol levels have a partially different genetic basis. We identified three specific genes responsible for high ethanol accumulation capacity, of which one gene encodes a protein kinase involved in DNA damage repair. Our work provides the first insight in the genetic basis of maximal ethanol accumulation capacity, shows that it involves different genetic elements compared to tolerance of cell proliferation to high ethanol levels, and reveals for the first time the importance of DNA damage repair in ethanol tolerance.
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.
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.
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.
DNA-protein cross-link; frameshift mutations; Complex insertions; DNA Polymerase ζ; DNA Polymerase η
Copy number expansions such as amplifications and duplications contribute to human phenotypic variation, promote molecular diversification during evolution, and drive the initiation and/or progression of various cancers. The mechanisms underlying these copy number changes are still incompletely understood, however. We recently demonstrated that transient, limited re-replication from a single origin in Saccharomyces cerevisiae efficiently induces segmental amplification of the re-replicated region. Structural analyses of such re-replication induced gene amplifications (RRIGA) suggested that RRIGA could provide a new mechanism for generating copy number variation by non-allelic homologous recombination (NAHR). Here we elucidate this new mechanism and provide insight into why it is so efficient. We establish that sequence homology is both necessary and sufficient for repetitive elements to participate in RRIGA and show that their recombination occurs by a single-strand annealing (SSA) mechanism. We also find that re-replication forks are prone to breakage, accounting for the widespread DNA damage associated with deregulation of replication proteins. These breaks appear to stimulate NAHR between re-replicated repeat sequences flanking a re-initiating replication origin. Our results support a RRIGA model where the expansion of a re-replication bubble beyond flanking homologous sequences followed by breakage at both forks in trans provides an ideal structural context for SSA–mediated NAHR to form a head-to-tail duplication. Given the remarkable efficiency of RRIGA, we suggest it may be an unappreciated contributor to copy number expansions in both disease and evolution.
Duplications and amplifications of chromosomal segments are frequently observed in eukaryotic genomes, including both normal and cancerous human genomes. These copy number variations contribute to the phenotypic variation upon which natural selection acts. For example, the amplification of genes whose excessive copy number facilitates uncontrolled cell division is often selected for during tumor development. Copy number variations can often arise when repetitive sequence elements, which are dispersed throughout eukaryotic genomes, undergo a rearrangement called non-allelic homologous recombination. Exactly how these rearrangements occur is poorly understood. Here, using budding yeast to model this class of copy number variation, we uncover a new and highly efficient mechanism by which these variations can be generated. The precipitating event is the aberrant re-initiation of DNA replication at a replication origin. Normally the hundreds to thousands of origins scattered throughout a eukaryotic genome are tightly controlled such that each is permitted to initiate only once per cell cycle. However, disruptions in these controls can allow origins to re-initiate, and we show how the resulting DNA re-replication structure can be readily converted into a tandem duplication via non-allelic homologous recombination. Hence, the re-initiation of DNA replication is a potential source of copy number variation both in disease and during evolution.
Chromosomal DNA must be in single-strand form for important transactions such as replication, transcription, and recombination to occur. The single-strand DNA (ssDNA) is more prone to damage than double-strand DNA (dsDNA), due to greater exposure of chemically reactive moieties in the nitrogenous bases. Thus, there can be agents that damage regions of ssDNA in vivo while being inert toward dsDNA. To assess the potential hazard posed by such agents, we devised an ssDNA–specific mutagenesis reporter system in budding yeast. The reporter strains bear the cdc13-1 temperature-sensitive mutation, such that shifting to 37°C results in telomere uncapping and ensuing 5′ to 3′ enzymatic resection. This exposes the reporter region, containing three closely-spaced reporter genes, as a long 3′ ssDNA overhang. We validated the ability of the system to detect mutagenic damage within ssDNA by expressing a modified human single-strand specific cytosine deaminase, APOBEC3G. APOBEC3G induced a high density of substitutions at cytosines in the ssDNA overhang strand, resulting in frequent, simultaneous inactivation of two reporter genes. We then examined the mutagenicity of sulfites, a class of reactive sulfur oxides to which humans are exposed frequently via respiration and food intake. Sulfites, at a concentration similar to that found in some foods, induced a high density of mutations, almost always as substitutions at cytosines in the ssDNA overhang strand, resulting in simultaneous inactivation of at least two reporter genes. Furthermore, sulfites formed a long-lived adducted 2′-deoxyuracil intermediate in DNA that was resistant to excision by uracil–DNA N-glycosylase. This intermediate was bypassed by error-prone translesion DNA synthesis, frequently involving Pol ζ, during repair synthesis. Our results suggest that sulfite-induced lesions in DNA can be particularly deleterious, since cells might not possess the means to repair or bypass such lesions accurately.
A cell's genome is encoded within double-strand DNA. Yet DNA must exist transiently in single-strand form to template transcription, replication, and repair. As DNA is more prone to damage in single-strand (ssDNA) than double-strand (dsDNA) form, there can be agents that mutate ssDNA, but not dsDNA. Since existing experimental systems cannot identify mutagens specifically mutating ssDNA inside cells, we devised a system of yeast strains, containing three closely-spaced reporter genes, for this purpose. We exposed yeast, under conditions where reporter DNA is single-stranded, either to an enzyme (APOBEC3G) or to a chemical (sulfites) that modifies cytosine, resulting in clusters of mutations that inactivated multiple reporter genes. Neither agent induced mutations in control strains where reporter genes remained double-stranded, confirming that both are potent ssDNA–specific mutagens within cells. Finally, our approach ascertained molecular mechanisms of action by which agents can mutate ssDNA specifically within cells, an area that warrants much investigation following reports that ssDNA–specific damage accounts for similar mutation clusters, and up to 40% of all mutations, in various cancers.
DNA amplification is a molecular process that increases the copy number of a chromosomal tract and often causes elevated expression of the amplified gene(s). Although gene amplification is frequently observed in cancer and other degenerative disorders, the molecular mechanisms involved in the process of DNA copy number increase remain largely unknown. We hypothesized that small DNA fragments could be the trigger of DNA amplification events. Following our findings that small fragments of DNA in the form of DNA oligonucleotides can be highly recombinogenic, we have developed a system in the yeast Saccharomyces cerevisiae to capture events of chromosomal DNA amplification initiated by small DNA fragments. Here we demonstrate that small DNAs can amplify a chromosomal region, generating either tandem duplications or acentric extrachromosomal DNA circles. Small fragment-driven DNA amplification (SFDA) occurs with a frequency that increases with the length of homology between the small DNAs and the target chromosomal regions. SFDA events are triggered even by small single-stranded molecules with as little as 20-nt homology with the genomic target. A double-strand break (DSB) external to the chromosomal amplicon region stimulates the amplification event up to a factor of 20 and favors formation of extrachromosomal circles. SFDA is dependent on Rad52 and Rad59, partially dependent on Rad1, Rad10, and Pol32, and independent of Rad51, suggesting a single-strand annealing mechanism. Our results reveal a novel molecular model for gene amplification, in which small DNA fragments drive DNA amplification and define the boundaries of the amplicon region. As DNA fragments are frequently found both inside cells and in the extracellular environment, such as the serum of patients with cancer or other degenerative disorders, we propose that SFDA may be a common mechanism for DNA amplification in cancer cells, as well as a more general cause of DNA copy number variation in nature.
DNA amplification is a copy-number increase of a DNA segment. Although DNA amplification is frequently observed in cancer and other degenerative disorders, the molecular mechanisms initiating this process are still largely elusive. Here we demonstrate that small DNA fragments with homology to two distant loci on the same chromosomal arm can trigger amplification of the region between the loci in yeast S. cerevisiae. Small fragment-driven DNA amplification (SFDA) is detected as intrachromosomal tandem duplications or extrachromosomal circles. Furthermore, a double-strand break several kilobases from the chromosomal amplicon region stimulates SFDA. SFDA efficiency depends on the homology length shared by the small DNAs and the target chromosomal loci. Homology as short as 20 nucleotides and even single-stranded molecules trigger SFDA. These results reveal a novel mechanism for initiating gene amplification, which could occur in cancer cells and could contribute to copy-number polymorphisms driving genetic variation in humans and other organisms.
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.
Polζ; Rev1; AP sites; mutagenesis; translesion synthesis
Homologous recombination is a universal mechanism that allows repair of DNA and provides support for DNA replication. Homologous recombination is therefore a major pathway that suppresses non-homology-mediated genome instability. Here, we report that recovery of impeded replication forks by homologous recombination is error-prone. Using a fork-arrest-based assay in fission yeast, we demonstrate that a single collapsed fork can cause mutations and large-scale genomic changes, including deletions and translocations. Fork-arrest-induced gross chromosomal rearrangements are mediated by inappropriate ectopic recombination events at the site of collapsed forks. Inverted repeats near the site of fork collapse stimulate large-scale genomic changes up to 1,500 times over spontaneous events. We also show that the high accuracy of DNA replication during S-phase is impaired by impediments to fork progression, since fork-arrest-induced mutation is due to erroneous DNA synthesis during recovery of replication forks. The mutations caused are small insertions/duplications between short tandem repeats (micro-homology) indicative of replication slippage. Our data establish that collapsed forks, but not stalled forks, recovered by homologous recombination are prone to replication slippage. The inaccuracy of DNA synthesis does not rely on PCNA ubiquitination or trans-lesion-synthesis DNA polymerases, and it is not counteracted by mismatch repair. We propose that deletions/insertions, mediated by micro-homology, leading to copy number variations during replication stress may arise by progression of error-prone replication forks restarted by homologous recombination.
The appropriate transmission of genetic material during successive cell divisions requires the accurate duplication and segregation of parental DNA. The semi-conservative replication of chromosomes during S-phase is highly accurate and prevents accumulation of deleterious mutations. However, during each round of duplication, there are many impediments to the replication fork machinery that may hinder faithful chromosome duplication. Homologous recombination is a universal mechanism involved in the rescue of replication forks by rebuilding a replication apparatus at the fork (by mechanisms that are not yet understood). However, recombination can jeopardize genome stability because it allows genetic exchanges between homologous repeated sequences dispersed through the genome. In this study, we employ a fission yeast-based arrest of a single replication fork to investigate the consequences of replication fork arrest for genome stability. We report that a single blocked fork favours genomic deletions, translocations, and mutations; and this instability occurs during fork recovery by recombination. We also report that a single arrested fork that resumes its progression by recombination is prone to causing replication slippage mediated by micro-homology. We propose that deletions/duplications observed in human cancer cells suffering from replication stress can be viewed as scars left by error-prone replication forks restarted by recombination.
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.
G4 DNA; R-loops; genome instability; transcription; recombination
Translesion synthesis (TLS) polymerases are specialized DNA polymerases capable of inserting nucleotides opposite DNA lesions that escape removal by dedicated DNA repair pathways. TLS polymerases allow cells to complete DNA replication in the presence of damage, thereby preventing checkpoint activation, genome instability, and cell death. Here, we characterize functional knockouts for polh-1 and polk-1, encoding the Caenorhabditis elegans homologs of the Y-family TLS polymerases η and κ. POLH-1 acts at many different DNA lesions as it protects cells against a wide range of DNA damaging agents, including UV, γ-irradiation, cisplatin, and methyl methane sulphonate (MMS). POLK-1 acts specifically but redundantly with POLH-1 in protection against methylation damage. Importantly, both polymerases play a prominent role early in embryonic development to allow fast replication of damaged genomes. Contrary to observations in mammalian cells, we show that neither POLH-1 nor POLK-1 is required for homologous recombination (HR) repair of DNA double-strand breaks. A genome-wide RNAi screen for genes that protect the C. elegans genome against MMS–induced DNA damage identified novel components in DNA damage bypass in the early embryo. Our data suggest SUMO-mediated regulation of both POLH-1 and POLK-1, and point towards a previously unrecognized role of the nuclear pore in regulating TLS.
Unrepaired DNA damage on the template strand poses a problem for the progression of the replication fork. Specialized translesion synthesis (TLS) polymerases are capable of bypassing DNA lesions without repairing them. Here, we use the nematode C. elegans, to show that there is modulation of the choice between repair and bypass during development. We show that during gametogenesis and later development repair dominates, while there is a short phase during embryonic development where resistance to damage depends heavily on TLS polymerases. The rapid divisions at this stage do not allow for delay in which repair processes can occur. Furthermore, we identify new factors that may play a role in the regulation of TLS during early embryogenesis.
DNA damage checkpoint activation can be subdivided in two steps: initial activation and signal amplification. The events distinguishing these two phases and their genetic determinants remain obscure. TopBP1, a mediator protein containing multiple BRCT domains, binds to and activates the ATR/ATRIP complex through its ATR-Activation Domain (AAD). We show that Schizosaccharomyces pombe Rad4TopBP1 AAD–defective strains are DNA damage sensitive during G1/S-phase, but not during G2. Using lacO-LacI tethering, we developed a DNA damage–independent assay for checkpoint activation that is Rad4TopBP1 AAD–dependent. In this assay, checkpoint activation requires histone H2A phosphorylation, the interaction between TopBP1 and the 9-1-1 complex, and is mediated by the phospho-binding activity of Crb253BP1. Consistent with a model where Rad4TopBP1 AAD–dependent checkpoint activation is ssDNA/RPA–independent and functions to amplify otherwise weak checkpoint signals, we demonstrate that the Rad4TopBP1 AAD is important for Chk1 phosphorylation when resection is limited in G2 by ablation of the resecting nuclease, Exo1. We also show that the Rad4TopBP1 AAD acts additively with a Rad9 AAD in G1/S phase but not G2. We propose that AAD–dependent Rad3ATR checkpoint amplification is particularly important when DNA resection is limiting. In S. pombe, this manifests in G1/S phase and relies on protein–chromatin interactions.
DNA structure–dependent checkpoint activation and the amplification of checkpoint signals are carefully modulated to allow the checkpoint kinases to delay mitosis and regulate DNA metabolism. While much work has gone into understanding how this checkpoint functions, the mechanism by which the checkpoint signal is amplified is less clear. We have characterised a conserved domain in the Schizosaccharomyces pombe TopBP1 homolog, Rad4TopBP1 (also known as Cut5) that is capable of activating the ATR homolog Rad3ATR. We demonstrate that this domain is not required for initial checkpoint activation, but functions to amplify the checkpoint signal, likely when the presence of single-stranded DNA is limiting. Our data suggest that the function of the Rad4TopBP1 ATR-Activation Domain (AAD) is mediated by interactions between checkpoint proteins and phosphorylated histone H2A, which is itself promoted by Rad3ATR. We propose that the resulting amplification of the checkpoint signal is particularly important in G1-S phase, when resection is limited.
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.
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.
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.
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.
Accurate estimates of mutation rates provide critical information to analyze genome evolution and organism fitness. We used whole-genome DNA sequencing, pulse-field gel electrophoresis, and comparative genome hybridization to determine mutation rates in diploid vegetative and meiotic mutation accumulation lines of Saccharomyces cerevisiae. The vegetative lines underwent only mitotic divisions while the meiotic lines underwent a meiotic cycle every ∼20 vegetative divisions. Similar base substitution rates were estimated for both lines. Given our experimental design, these measures indicated that the meiotic mutation rate is within the range of being equal to zero to being 55-fold higher than the vegetative rate. Mutations detected in vegetative lines were all heterozygous while those in meiotic lines were homozygous. A quantitative analysis of intra-tetrad mating events in the meiotic lines showed that inter-spore mating is primarily responsible for rapidly fixing mutations to homozygosity as well as for removing mutations. We did not observe 1–2 nt insertion/deletion (in-del) mutations in any of the sequenced lines and only one structural variant in a non-telomeric location was found. However, a large number of structural variations in subtelomeric sequences were seen in both vegetative and meiotic lines that did not affect viability. Our results indicate that the diploid yeast nuclear genome is remarkably stable during the vegetative and meiotic cell cycles and support the hypothesis that peripheral regions of chromosomes are more dynamic than gene-rich central sections where structural rearrangements could be deleterious. This work also provides an improved estimate for the mutational load carried by diploid organisms.
Mutations result from errors that occur during DNA metabolism. They provide the raw materials for evolution, can affect organism fitness, and have been shown to accumulate in organisms during asexual growth. During a sexual life cycle, mutations can be removed by recombination and mating. While such removal is thought to provide a fitness advantage, studies have shown that recombination itself is mutagenic. To examine if the mutation rate in an organism differs during asexual and sexual cycles, we sequenced the entire nuclear genome of lines of diploid baker's yeast that underwent only asexual growth, or alternating cycles of asexual and sexual growth. The estimated rate of base substitutions in the vegetative lines was extremely low (2.9×10−10 base substitutions per base per cell generation) and the meiotic mutation rate is within the range of being equal to zero to being 55 times higher than the vegetative rate. Interestingly, we observed a large number of changes in the ends of chromosomes in the asexual and sexual cycles that did not affect fitness; changes at other locations were very rare, suggesting a remarkable genome stability of diploid baker's yeast.
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.
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.
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.
spontaneous mutagenesis; transcription; replication direction; polymerase zeta; mutation spectrum
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.