Mitotic Gene Conversion Tracts Associated with Repair of a Defined Double-Strand Break in Saccharomyces cerevisiae

Abstract

Mitotic recombination between homologous chromosomes leads to the uncovering of recessive alleles through loss of heterozygosity. In the current study, a defined double-strand break was used to initiate reciprocal loss of heterozygosity between diverged homologs of chromosome IV in These events resulted from the repair of two broken chromatids, one of which was repaired as a crossover and the other as a noncrossover. Associated gene conversion tracts resulting from the donor-directed repair of mismatches formed during strand exchange (heteroduplex DNA) were mapped using microarrays. Gene conversion tracts associated with individual crossover and noncrossover events were similar in size and position, with half of the tracts being unidirectional and mapping to only one side of the initiating break. Among crossover events, this likely reflected gene conversion on only one side of the break, with restoration-type repair occurring on the other side. For noncrossover events, an ectopic system was used to directly compare gene conversion tracts produced in a wild-type strain to heteroduplex DNA tracts generated in the absence of the Mlh1 mismatch-repair protein. There was a strong bias for unidirectional tracts in the absence, but not in the presence, of Mlh1 This suggests that mismatch repair acts on heteroduplex DNA that is only transiently present in noncrossover intermediates of the synthesis dependent strand annealing pathway. Although the molecular features of events associated with loss of heterozygosity generally agreed with those predicted by current recombination models, there were unexpected complexities in associated gene conversion tracts.

DNA strand-exchange patterns associated with double-strand break-induced and spontaneous mitotic crossovers in Saccharomyces cerevisiae

“…Though the length of homology on each side of the induced DSB was similar in the two systems (~2 kb), the median length of CO-associated hetDNA was ~1.9 kb, while that associated with SDSA events was only ~1.1 kb (p = 0.0002). The significant hetDNA length difference between COs and NCOs in similar ectopic systems, but not in allelic systems [ 43 , 45 ], is consistent with our previous suggestion that the second-end engagement, which is a prerequisite for reciprocal CO formation in the canonical DSB repair pathway, requires more extensive homology length than does SDSA [ 36 , 53 ]. We suggested that the D-loop size and stability is directly related to the length of hetDNA that forms during the initial strand invasion, which may be limited by substrate size in ectopic assays.…”

“…A similar event in MMR-proficient cells would give rise to a single gene conversion tract located on only one side of the DSB. We previously reported frequent DSB-initiated allelic CO events with just a single gene conversion tract and attributed these to frequent restoration-type repair [ 43 ]. Because D-loop migration erases the hetDNA created by strand invasion, however, this may have contributed to the one-sided gene conversion tracts.…”

Rapid Phenotypic and Genotypic Diversification After Exposure to the Oral Host Niche in Candida albicans

“…Furthermore, more than one mismatch machinery may have been involved in repairing breaks. Strikingly, similar LOH signatures were observed during mitotic DSB repair in S. cerevisiae (Guo et al 2017;Hum and Jinks-Robertson 2017), suggesting that these mechanisms may have been conserved through evolution.…”

Genome-wide analysis of genomic alterations induced by oxidative DNA damage in yeast

“…There are two plausible interpretations of the observation of the results shown in Figure 7A: oxidative damage during aerobic growth is responsible for about half of spontaneous recombination events, or recombinogenic lesions are less efficiently repaired in cells grown anaerobically. To distinguish between these possibilities, we examined the efficiency of repair of a DSB induced in a strain (SJR4317) with the galactose-inducible gene encoding the mega-endonuclease I-SceI (40) under aerobic and anaerobic conditions. This diploid is closely related to JSC25-1 described above, and contains the heterozygous SUP4-o marker near the end of the right arm of IV.…”