DAMAGE-INDUCED LOCALIZED HYPERMUTABILITY (LHM). Mutations are important in evolution as well as many diseases. We found that lesions in transient single-strand DNA (ssDNA) are especially threatening to genome stability and lead to clusters of multiple mutations. Continuing our previous studies of LHM we found that mutation clusters can occur in multiple types of human cancers. In agreement with findings in yeast, clusters were often found in the vicinity of rearrangement breakpoints. Strand-coordinated clusters of mutated cytosines or guanines were highly enriched with a motif targeted by APOBEC family of ssDNA-specific cytosine-deaminases involved in the innate immunity against viruses. In conjunction with the Broad Institute, Drs. Gordenin and Roberts participated in the analysis of mutation spectra in exome sequences from 3,083 tumor-normal pairs, primarily from The Cancer Genome Atlas (TCGA). The outcome was a discovery of wide variation in mutation frequency and spectrum within cancer types, shedding light on mutational processes and disease etiology. Importantly, a TC mutation signature was discovered that was reminiscent of APOBEC in several cancer types. Guided by this observation we developed analytical approaches for evaluating the strength of an APOBEC mutation pattern in the individual samples from multiple whole-genome and exome mutation datasets such as TCGA. Our approach to the statistical exploration of complex mutation spectra in multiple cancer samples involved formulation of a single hypothesis surrounding a diagnostic mutation pattern that would be based on information from prior experiments and data analyses. Enrichment for APOBEC signature mutations was calculated over the presence of the APOBEC mutation motif (TCW or WGA) in the +/- 20 nucleotides surrounding mutated nucleotides. We utilized only the immediate context surrounding mutations because APOBEC enzymes are thought to scan a limited area of ssDNA to deaminate C in a preferred sequence context. This approach does not exclude any given region of the genome, but rather utilizes regions where mutagenesis has occurred and then determines if the mutagenesis is enriched for the APOBEC signature. The APOBEC mutation pattern is prominent and even prevailing in many samples among several cancer types, as opposed to other cancer types where it is hardly detectable. Additionally, the appearance of APOBEC mutations correlates with APOBEC mRNA levels and extends into a subset of genes considered by multiple criteria to be cancer drivers. In order to assess the potential hazard posed by environmental agents to chromosomal ssDNA, we devised a ssDNA-specific mutagenesis reporter system in budding yeast. The reporter strains bear the cdc13-1 temperature sensitive mutation, such that shifting to 37oC results in telomere uncapping and ensuing 5 to 3 resection. The resection results in long ssDNA regions containing 3 closely-spaced reporter genes. We characterized the ssDNA mutagenic action of sulfites, a class of reactive sulfur oxides to which humans are exposed frequently. We found that sulfites form a long-lived adducted 2-deoxyuracil intermediate in DNA that is resistant to excision by uracil-DNA N-glycosylase and must be bypassed during repair synthesis by a translesion synthesis polymerase, most frequently Pol zeta, during repair synthesis. Our results suggest that sulfite-induced lesions in ssDNA can be particularly deleterious, since cells do not possess the means to repair or bypass such lesions accurately. In addition, this system provides an opportunity to address the relevance of single-strand DNA to genome stability when challenged by potential mutagens. We examined the impact of ssDNA that can arise as gaps during excision repair and possible associations with recombination following UV-exposure. Using our pulsed-field gel electrophoresis approaches for detecting very slow-moving DNA repair intermediates (SMD) and real-time monitoring of sister-chromatid recombination in a circular chromosome, we studied the gap filling process after UV damage, induced recombination and coordination of repair pathways. The amount of SMD and the time required for resolution was increased in mutants lacking TLS polymerases (Pol-eta and Pol-zeta) and recombination was required for UV repair in the absence of TLS. Thus, UV can induce recombination in the nonreplicating G2 stage and is dramatically increased with defects in gap filling process. The 5'to 3'Exo1, which provides excision dependent gap extension, is required for recombination repair. Moreover, the UV-induced recombination was facilitated by the topoisomerase Top3, which we propose assists the strand invasion process that is upstream of Rad51 and Rad52. Collectively, these results suggest a novel mechanism of recombination and reveal a complex and highly-coordinated repair profile of the ssDNA gap. EFFECTS OF COHESIN DEFECTS ON GENOME STABILITY. Gain or loss of chromosomes resulting in aneuploidy or loss-of heterozygosity (LOH) is an important factor in cancer and adaptive evolution. Although chromosome gain is often found in tumors and fungi and is a frequent event in all kingdoms, its genetic control has hardly been studied. We measured the rates of chromosome gain in WT yeast and sister chromatid cohesion (SCC) compromised strains. Even mild deactivation of the cohesion complex caused a high rate of chromosome gain. On top of defects in SCC, yeast cell type had a significant contribution to chromosome gain, with the greatest rates observed for homozygous mating type diploids, then heterozygous mating type and smallest in haploids under all SCC defective strains. We suggest that while chromosome gain due to SCC malfunction can have negative effects through gene imbalance it could also facilitate opportunities for evolution and adaptive changes. In multicellular organisms, both factors could lead to somatic diseases including cancer. The sister chromatid cohesion process (SCC) mediated via the cohesin complex tethers the newly replicated sister chromatids until mitosis and thus suppresses LOH through chromosome loss. SCC is also important to channel sister chromatid recombinational repair to sisters thereby preventing allelic recombination. Until our work, it was unknown how different mutations in the SCC pathway prevent different modes of LOH. We found that the cohesin mutation mcd1-1 and other mutations in SCC differentially affected various types of LOH of a centromeric marker. The effects of mutations were greatest for whole chromosome loss. We suggest that SCC malfunction could lead to extensive LOH and thus facilitate opportunities for evolution and carcinogenesis through homozygosis of many recessive mutations.

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Covo, Shay; Puccia, Christopher M; Argueso, Juan Lucas et al. (2014) The sister chromatid cohesion pathway suppresses multiple chromosome gain and chromosome amplification. Genetics 196:373-84
Sakofsky, Cynthia J; Roberts, Steven A; Malc, Ewa et al. (2014) Break-induced replication is a source of mutation clusters underlying kataegis. Cell Rep 7:1640-8
Roberts, Steven A; Gordenin, Dmitry A (2014) Clustered and genome-wide transient mutagenesis in human cancers: Hypermutation without permanent mutators or loss of fitness. Bioessays :
Witwer, Kenneth W; Hirschi, Kendal D (2014) Transfer and functional consequences of dietary microRNAs in vertebrates: concepts in search of corroboration: negative results challenge the hypothesis that dietary xenomiRs cross the gut and regulate genes in ingesting vertebrates, but important questio Bioessays 36:394-406
Lawrence, Michael S; Stojanov, Petar; Polak, Paz et al. (2013) Mutational heterogeneity in cancer and the search for new cancer-associated genes. Nature 499:214-8
Roberts, Steven A; Lawrence, Michael S; Klimczak, Leszek J et al. (2013) An APOBEC cytidine deaminase mutagenesis pattern is widespread in human cancers. Nat Genet 45:970-6
Degtyareva, Natalya P; Heyburn, Lanier; Sterling, Joan et al. (2013) Oxidative stress-induced mutagenesis in single-strand DNA occurs primarily at cytosines and is DNA polymerase zeta-dependent only for adenines and guanines. Nucleic Acids Res 41:8995-9005
Ma, Wenjian; Westmoreland, James W; Resnick, Michael A (2013) Homologous recombination rescues ssDNA gaps generated by nucleotide excision repair and reduced translesion DNA synthesis in yeast G2 cells. Proc Natl Acad Sci U S A 110:E2895-904
Covo, Shay; Westmoreland, James W; Reddy, Amit K et al. (2012) RAD53 is limiting in double-strand break repair and in protection against toxicity associated with ribonucleotide reductase inhibition. DNA Repair (Amst) 11:317-23
Chan, Kin; Sterling, Joan F; Roberts, Steven A et al. (2012) Base damage within single-strand DNA underlies in vivo hypermutability induced by a ubiquitous environmental agent. PLoS Genet 8:e1003149

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