High-Resolution Mapping of Spontaneous Mitotic Recombination Hotspots on the 1.1 Mb Arm of Yeast Chromosome IV

Abstract

Although homologous recombination is an important pathway for the repair of double-stranded DNA breaks in mitotically dividing eukaryotic cells, these events can also have negative consequences, such as loss of heterozygosity (LOH) of deleterious mutations. We mapped about 140 spontaneous reciprocal crossovers on the right arm of the yeast chromosome IV using single-nucleotide-polymorphism (SNP) microarrays. Our mapping and subsequent experiments demonstrate that inverted repeats of Ty retrotransposable elements are mitotic recombination hotspots. We found that the mitotic recombination maps on the two homologs were substantially different and were unrelated to meiotic recombination maps. Additionally, about 70% of the DNA lesions that result in LOH are likely generated during G1 of the cell cycle and repaired during S or G2. We also show that different genetic elements are associated with reciprocal crossover conversion tracts depending on the cell cycle timing of the initiating DSB.

Reconstitution of long and short patch mismatch repair reactions using Saccharomyces cerevisiae proteins

“…This excision reaction required Exo1 and was stimulated by both a mispair and Msh2-Msh6, where mispair binding by Msh2-Msh6 was required for stimulating excision. Strikingly, the excision tracts observed were very long, as long as 1,750 bp in reactions with the substrate containing a nick at the NaeI site and 2,900 bp with the substrate containing a nick at the AflIII site, which approximates the length of MMR excision tracts predicted from genetic studies of gene conversion (12,53). The electron microscopy experiments indicated that the length of the longest excision tract was threefold longer in the presence of Msh2-Msh6.…”

Genome-wide high-resolution mapping of chromosome fragile sites in Saccharomyces cerevisiae

“…In our previous studies of spontaneous (22) and UV-induced mitotic recombination events (23), more than half of the crossover-associated gene-conversion events were 4:0 conversions (Fig. 5C) or 3:1/4:0 hybrid events (Fig.…”

“…In diploid strains that are heterozygous for many SNPs, mitotic recombination events between homologous chromosomes can be mapped by identifying regions that have undergone loss of heterozygosity (LOH). Previously, using SNP-specific microarrays (21), we mapped spontaneous recombination events on the right arm of chromosome IV (22), and UV-induced events throughout the genome (23). In our current study, we map unselected mitotic recombination events induced by low levels of DNA polymerase-α throughout the yeast genome, in addition to mapping reciprocal cross-overs selected on chromosomes V and IV.…”

“…To detect recombination events for the entire yeast genome, we used oligonucleotide-containing microarrays (20,22). Each individual heterozygous SNP that was assayed was represented by four 25-base oligonucleotides, two that perfectly matched the Watson and Crick strands of the W303a-specific SNP and two that perfectly matched the Watson and Crick strands of the YJM789-specific SNP.…”

“…The experiments described above did not allow us to distinguish cross-overs and BIR events, nor to examine the types of gene conversions associated with cross-overs. For this analysis, we used diploid strains in which cross-overs could be detected by a sectoring assay (22,23,26). In these strains, one homolog has the ochre-suppressor SUP4-o located near the telomere and the hygromycin-resistant HYG gene located at a similar position on the other homolog; in addition, the diploid is homozygous for ade2-1, an ochre mutation that, when unsuppressed, results in red colonies.…”