Summary Mutations are typically perceived as random, independent events. We describe here non-random clustered mutations in yeast and in human cancers. Genome sequencing of yeast grown under chronic alkylation damage identified mutation clusters that extend up to 200 kb. A predominance of “strand-coordinated” changes of either cytosines or guanines in the same strand, mutation patterns and genetic controls indicated that simultaneous mutations were generated by base alkylation in abnormally long single-strand (ss)DNA formed at double-strand breaks (DSBs) and replication forks. Significantly, we found mutation clusters with analogous features in sequenced human cancers. Strand-coordinated clusters of mutated cytosines or guanines often resided near chromosome rearrangement breakpoints and were highly enriched with a motif targeted by APOBEC family cytosine-deaminases, which strongly prefer ssDNA. These data indicate that hyper-mutation via multiple simultaneous changes in randomly formed ssDNA is a general phenomenon that may be an important mechanism producing rapid genetic variation.

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The major DNA repair pathways operate on damage in double-strand DNA because they use the intact strand as a template after damage removal. Therefore, lesions in transient single-strand stretches of chromosomal DNA are expected to be especially threatening to genome stability. To test this hypothesis, we designed systems in budding yeast that could generate many kilobases of persistent single-strand DNA next to double-strand breaks or uncapped telomeres. The systems allowed controlled restoration to the double-strand state after applying DNA damage. We found that lesions induced by UV-light and methyl methanesulfonate can be tolerated in long single-strand regions and are hypermutagenic. The hypermutability required PCNA monoubiquitination and was largely attributable to translesion synthesis by the error-prone DNA polymerase ζ. In support of multiple lesions in single-strand DNA being a source of hypermutability, analysis of the UV-induced mutants revealed strong strand-specific bias and unexpectedly high frequency of alleles with widely separated multiple mutations scattered over several kilobases. Hypermutability and multiple mutations associated with lesions in transient stretches of long single-strand DNA may be a source of carcinogenesis and provide selective advantage in adaptive 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.

Okazaki fragment maturation to produce continuous lagging strands in eukaryotic cells requires precise coordination of strand displacement synthesis by DNA polymerase ␦ (Pol ␦) with 5-flap cutting by FEN1 RAD27 endonuclease. Excessive strand displacement is normally prevented by the 3-exonuclease activity of Pol ␦. This core maturation machinery can be assisted by Dna2 nuclease/helicase that processes long flaps. Our genetic studies show that deletion of the POL32 (third subunit of Pol ␦) or PIF1 helicase genes can suppress lethality or growth defects of rad27⌬ pol3-D520V mutants (defective for FEN1 RAD27 and the 3-exonuclease of Pol ␦) that produce long flaps and of dna2⌬ mutants that are defective in cutting long flaps. On the contrary, pol32⌬ or pif1⌬ caused lethality of rad27⌬ exo1⌬ double mutants, suggesting that Pol32 and Pif1 are required to generate longer flaps that can be processed by Dna2 in the absence of the short flap processing activities of FEN1 RAD27 and Exo1. The genetic analysis reveals a remarkable flexibility of the Okazaki maturation machinery and is in accord with our biochemical analysis. In vitro, the generation of short flaps by Pol ␦ is not affected by the presence of Pol32; however, longer flaps only accumulate when Pol32 is present. The presence of FEN1 RAD27 during strand displacement synthesis curtails displacement in favor of flap cutting, thus suggesting an active hand-off mechanism from Pol ␦ to FEN1 RAD27 . Finally, RNA-DNA hybrids are more readily displaced by Pol ␦ than DNA hybrids, thereby favoring degradation of initiator RNA during Okazaki maturation.The process of DNA replication in eukaryotic cells leads to the generation of a vast number of Okazaki fragments on the lagging strand of the replication fork. Approximately 50,000,000 Okazaki fragments are synthesized when a human cell replicates, and all of these need to be efficiently and accurately matured into continuous lagging strands to ensure genome integrity. Various DNA structures are generated during the synthesis and maturation of Okazaki fragments. These structures constitute the largest pool for potential DNA damage in the cell. Incomplete or poorly processed Okazaki fragments can lead to repeat expansion mutations, small duplication mutations, and to the generation of double-stranded DNA breaks (1).A large number of activities have been implicated in lagging strand DNA maturation. In the budding yeast Saccharomyces cerevisiae, the RAD27 gene product is the 5Ј-flap endonuclease FEN1. FEN1RAD27 has been assigned a dominant role in creating ligatable nicks during Okazaki maturation (reviewed in Refs. 2 and 3). Strong support for the importance of RAD27 in Okazaki maturation was initially provided by the dramatic increase of small duplications up to ϳ100 nt 4 in length flanked by short repeats in rad27-null mutants (4). This unusual class of duplication mutations was proposed to result through ligation of an unremoved flap with the 3Ј-end of the downstream Okazaki fragment. This type of duplications is caused not o...

Until recently, the only biological function attributed to the 335 exonuclease activity of DNA polymerases was proofreading of replication errors. Based on genetic and biochemical analysis of the 335 exonuclease of yeast DNA polymerase ␦ (Pol ␦) we have discerned additional biological roles for this exonuclease in Okazaki fragment maturation and mismatch repair. We asked whether Pol ␦ exonuclease performs all these biological functions in association with the replicative complex or as an exonuclease separate from the replicating holoenzyme. We have identified yeast Pol ␦ mutants at Leu523 that are defective in processive DNA synthesis when the rate of misincorporation is high because of a deoxynucleoside triphosphate (dNTP) imbalance. Yet the mutants retain robust 335 exonuclease activity. Based on biochemical studies, the mutant enzymes appear to be impaired in switching of the nascent 3 end between the polymerase and the exonuclease sites, resulting in severely impaired biological functions. Mutation rates and spectra and synergistic interactions of the pol3-L523X mutations with msh2, exo1, and rad27/fen1 defects were indistinguishable from those observed with previously studied exonuclease-defective mutants of the Pol ␦. We conclude that the three biological functions of the 335 exonuclease addressed in this study are performed intramolecularly within the replicating holoenzyme.DNA replication errors are an important source of genetic change. Several biochemical activities have evolved in order to prevent errors from becoming mutations. One is the 3Ј35Ј exonuclease (Exo) activity present in many DNA polymerases (Pol). A well-established function for this exonuclease activity is the proofreading of errors made by the DNA polymerase (6, 27). Mismatch repair (MMR) is a second fidelity system that can correct replication errors which escape proofreading. Characteristically, mutations that inactivate the exonuclease activity of the replicative DNA polymerase combined with those which inactivate MMR confer a very strong mutator phenotype, far exceeding the sum of individual mutator effects. Such synergistic hypermutability caused by a combination of proofreading and MMR defects can also lead to accumulation of lethal mutations sufficient to block propagation of a doublemutant strain (31,35).Another synergistic interaction between exonuclease deficiency in Pol ␦ and mutations in RAD27, which encodes 5Ј-flap endonuclease FEN1, highlights the role for the Pol ␦-Exo in creating or maintaining a ligatable nick during Okazaki fragment maturation. These two genetic defects are often synthetic lethal (15,25). In vivo evidence that Pol ␦-Exo supplements FEN1 in Okazaki maturation was obtained with viable double mutants that that involved a rad27-p allele with a partial defect. These mutants exhibited hyperrecombination and an unusual pattern of hypermutability. The most frequent class of mutations were extended duplications (up to 100 bp) flanked by short direct repeats (4 to 10 bp) (24). Such mutations are usually observed...

Localized hyper-mutability caused by accumulation of lesions in persistent single-stranded (ss) DNA has been recently found in several types of cancers. An increase in endogenous levels of reactive oxygen species (ROS) is considered to be one of the hallmarks of cancers. Employing a yeast model system, we addressed the role of oxidative stress as a potential source of hyper-mutability in ssDNA by modulation of the endogenous ROS levels and by exposing cells to oxidative DNA-damaging agents. We report here that under oxidative stress conditions the majority of base substitution mutations in ssDNA are caused by erroneous, DNA polymerase (Pol) zeta-independent bypass of cytosines, resulting in C to T transitions. For all other DNA bases Pol zeta is essential for ROS-induced mutagenesis. The density of ROS-induced mutations in ssDNA is lower, compared to that caused by UV and MMS, which suggests that ssDNA could be actively protected from oxidative damage. These findings have important implications for understanding mechanisms of oxidative mutagenesis, and could be applied to development of anticancer therapies and cancer prevention.

Variations in mutation rates across the genome have been demonstrated both in model organisms and in cancers. This phenomenon is largely driven by the damage specificity of diverse mutagens and the differences in DNA repair efficiency in given genomic contexts. Here, we demonstrate that the single-strand DNA-specific cytidine deaminase APOBEC3B (A3B) damages tRNA genes at a 1000-fold higher efficiency than other non-tRNA genomic regions in budding yeast. We found that A3B-induced lesions in tRNA genes were predominantly located on the non-transcribed strand, while no transcriptional strand bias was observed in protein coding genes. Furthermore, tRNA gene mutations were exacerbated in cells where RNaseH expression was completely abolished (Δrnh1Δrnh35). These data suggest a transcription-dependent mechanism for A3B-induced tRNA gene hypermutation. Interestingly, in strains proficient in DNA repair, only 1% of the abasic sites formed upon excision of A3B-deaminated cytosines were not repaired leading to mutations in tRNA genes, while 18% of these lesions failed to be repaired in the remainder of the genome. A3B-induced mutagenesis in tRNA genes was found to be efficiently suppressed by the redundant activities of both base excision repair (BER) and the error-free DNA damage bypass pathway. On the other hand, deficiencies in BER did not have a profound effect on A3B-induced mutations in CAN1, the reporter for protein coding genes. We hypothesize that differences in the mechanisms underlying ssDNA formation at tRNA genes and other genomic loci are the key determinants of the choice of the repair pathways and consequently the efficiency of DNA damage repair in these regions. Overall, our results indicate that tRNA genes are highly susceptible to ssDNA-specific DNA damaging agents. However, increased DNA repair efficacy in tRNA genes can prevent their hypermutation and maintain both genome and proteome homeostasis.