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 MMS, which causes random DNA damage. A novel mutation reporter was developed for selecting forward mutations in URA3 and CAN1. Chronic exposure to MMS caused joint inactivation of URA3 and CAN1 when they were close (separated by 1 kb) but not when they were separated by 85 kb, indicating that the double mutations occurred primarily via a single localized event. To identify mutation clusters and underlying mechanisms, we improved whole genome sequencing 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 have 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. From our preliminary analysis of mutations found by whole-genome sequencing in several dozens of different tumors we conclude that clusters of simultaneous mutations occurred among a vast number of base substitutions. DSBS RESULTING FROM DNA BASE DAMAGE. Our studies on MMS have also been extended to mechanisms by which MMS can cause derived DSBs. Little is known about derived DSBs, which might arise during normal or defective base excision repair (BER) of single-strand lesions. We employed our recent approaches for detecting MMS-induced DSBs (MCB 2009) and end-processing (resection) of ionizing radiation-induced DSBs (PLoS Genetics, 2009) to address the role of BER in generation of derived DSBs and mechanisms of repair. Previously, we found that MMS damage, which does not cause single strand breaks directly, led to accumulation of unrepairable DSBs in G1 haploid yeast that lack AP endonucleases (Apn1/2). We have recently shown that the MMS-derived DSBs in G2 cells could be repaired via the RAD52 pathway utilizing homologous recombination between sister chromatids. Although the ends are dirty, we find that they are efficiently resected as part of the recombinational repair process. Interestingly, using pulsed-field gel electrophoresis (PFGE) of chromosomal DNA we identified a very slow-moving DNA intermediate (SMD). The SMD was quickly formed after short MMS exposure and was present up to 4 hr later. It is unlikely due to recombination since RAD50 or RAD51 mutants still present SMD. To address the origin and resection of the derived DSBs, we blocked either the first step in BER, formation of AP sites, by deletion of the MAG1 glycosylase or the following step, cleavage of AP sites, by methoxyamine treatment of cells. Since the MMS derived DSBs as well as SMD were completely blocked, they must arise through the processing of AP sites. However, additional deletion of NTG1 and NTG2 lyases (i.e., apn1/2 ntg1/2) which are involved in the alternative pathway for processing AP sites, reduced the amount of DSBs but not SMD, suggesting that SMD does not arise during the generation or processing of DSBs. Collectively, these results reveal a complex repair profile of base lesions in the absence of AP endonucleases. Our results highlight the pathway for generation, processing and repair of DSBs caused by base alkylation. GENE DOSAGE OF GENOME STABILITY GENES. The sister chromatid cohesion (SCC) complex known as cohesin assures close association between sister chromatids and faithful transmission of chromosomes in mitosis. Cohesin is also involved in chromosome structure, maintenance, transcription DNA repair, and cohesin mutations are associated with cancer and developmental defects. Using a gene dosage approach developed in tetraploid yeast we reported (PLoS Genetics, 2010) that cohesin is present in limiting amounts and that reduced levels greatly enhance the risk of DNA damage-induced genome instability in G2 cells. Given the importance of cohesin levels in genome stability, we have extended our initial findings about cohesion gene dosage into addressing 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 lead to increases in BIR and chromosome loss up to 50-fold higher over WT. There were also unexpected consequences of such misrouting: chromosome nondisjunction and amplification. 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. Based on our results, 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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