DAMAGE-INDUCED LOCALIZED HYPERMUTABILITY (LHM). Mutations are important in evolution as well as many diseases. While mutations are generally considered to accumulate independently, most single base substitutions in coding sequences fail to significantly alter the activity of the corresponding protein. Multiple mutations may be needed to produce dramatic genetic consequences such as gene inactivation or generation of alleles with novel function. 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 yeast grown in the presence of methylmethane sulfonate (MMS). Chronic exposure to MMS caused joint inactivation of the forward mutation reporters URA3 and CAN1 when they were close (separated by 1 kb) but not when they were separated by 85 kb, indicating double mutations occurred primarily via a single localized event. Whole genome sequencing was improved to a level where single base substitutions in 85% of the yeast genome could be detected (collaboration with Dr. Piotr Mieczkowski). Surprisingly, inactivation of URA3 and CAN1 is often accompanied by additional mutations (up to 30) in clusters that span up to 250 kb. The cluster densities were as high as 1/kb. Unlike mutations in the rest of the genome, clusters were predominantly composed of mutations of G:C pairs and contained a strand bias consistent with the mutation spectra of error-prone TLS occurring during restoration of MMS-damaged ssDNA. This base specificity and strand bias indicates DSB associated strand-resection as a major pathway for the LHM in wild type cells. We have also identified a second pathway where mutation clusters occur in ssDNA generated in the mutant cell lacking tof1/timeless-csm3/tipin replication fork protection complex. These smaller clusters (spanning only a few kB) likely stem from broken or uncoupled replication forks. Thus, we identified two pathways of damage-induced mutagenesis in which the combination of localized inability to repair DNA damage along with error-prone translesion synthesis leads to localized severe genetic alteration within a single generation. This scenario could result in rapid diversification and selective advantage in adaptive evolution. It also identifies a possible new source of genetic disease and cancer. Our analysis of mutations found by whole-genome sequencing in several dozens of different tumors have revealed clusters of simultaneous mutations in three types of human cancers, multiple myelomas, prostate carcinomas and in head and neck squamous cells carcinomas. 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. 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 in cancers as well as in normal somatic tissues. 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. GENE DOSAGE OF GENOME STABILITY GENES. The sister chromatid cohesion (SCC) complex is involved in chromosome transmission, chromosome structure, maintenance, transcription DNA repair, and cohesin mutations are associated with cancer and developmental defects. We have extended initial findings about cohesin gene dosage to address the role of regulators and the mode of establishment of SCC in genome stability. We explored the cohesin complex per se (Mcd1) and its regulator Wpl1 and found that they prevent misrouting of recombinational DSB repair into break-induced replication (BIR). Haploid and diploid yeast carrying a deletion of WPL1 or a temperature-sensitive mutation mcd1-1 in an essential cohesin subunit have increased BIR and chromosome loss over WT. The mcd1-1 or wpl1 deletion diploids exhibited a dramatic increase (up to 1000-fold) in chromosomal nondisjunction and amplification, resulting in cells with 4 to 5 copies of the reporter chromosome. We propose that the SCC maintenance complex (Wpl1) prevents chromosome instability caused by breakage primarily through limiting BIR, while the core cohesin complex maintains chromosome stability by keeping nondisjunction of unbroken chromosomes as well as BIR at low levels. Using a tetraploid gene dosage model in which only one copy of the yeast RAD53 is functional (simplex), we found that the simplex strain was not sensitive to acute UV radiation or chronic MMS exposure. However, the simplex strain was sensitized to chronic exposure of the ribonucleotide reductase inhibitor hydroxyurea (HU). The importance of this finding is stressed by the fact that the Rad53, the homolog of human Chk2, is a central component of the DNA damage checkpoint system. Surprisingly, reduced RAD53 gene dosage did not affect sensitivity to HU acute exposure, indicating that immediate checkpoint responses and recovery from HU-induced stress were not compromised. We propose that a modest reduction in Rad53 activity can impact the activation of the ribonucleotide reductase catalytic subunit Rnr1 following stress, reducing the ability to generate nucleotide pools sufficient for DNA repair and replication.

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Sakofsky, Cynthia J; Roberts, Steven A; Malc, Ewa et al. (2014) Break-induced replication is a source of mutation clusters underlying kataegis. Cell Rep 7:1640-1648
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 :
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
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
Lawrence, Michael S; Stojanov, Petar; Polak, Paz et al. (2013) Mutational heterogeneity in cancer and the search for new cancer-associated genes. Nature 499:214-218
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
Covo, Shay; Ma, Wenjian; Westmoreland, James W et al. (2012) Understanding the origins of UV-induced recombination through manipulation of sister chromatid cohesion. Cell Cycle 11:3937-44
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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