Requirement of POL3 and POL4 on non-homologous and microhomology-mediated end joining in rad50/xrs2 mutants of Saccharomyces cerevisiae

Non-homologous end joining (NHEJ) directly joins two broken DNA ends without sequence homology. A distinct pathway called microhomology-mediated end joining (MMEJ) relies on a few base pairs of homology between the recombined DNA. The majority of DNA double-strand breaks caused by endogenous oxygen species or ionizing radiation contain damaged bases that hinder direct religation. End processing is required to remove mismatched nucleotides and fill in gaps during end joining of incompatible ends. POL3 in Saccharomyces cerevisiae encodes polymerase δ that is required for DNA replication and other DNA repair processes. Our previous results have shown that POL3 is involved in gap filling at 3′ overhangs in POL4-independent NHEJ. Here, we studied the epistatic interaction between POL3, RAD50, XRS2 and POL4 in NHEJ using a plasmid-based endjoining assay in yeast. We demonstrated that either rad50 or xrs2 mutation is epistatic for end joining of compatible ends in the rad50 pol3-t or xrs2 pol3-t double mutants. However, the pol3-t and rad50 or pol3-t and xrs2 mutants caused an additive decrease in the end-joining efficiency of incompatible ends, suggesting that POL3 and RAD50 or POL3 and XRS2 exhibit independent functions in NHEJ. In the rad50 pol4 mutant, end joining of incompatible ends was not detected. In the rad50 or xrs2 mutants, NHEJ events did not contain any microhomology at the rejoined junctions. The pol3-t mutation restored MMEJ in the rad50 or xrs2 mutant backgrounds. Moreover, we demonstrated that NHEJ of incompatible ends required RAD50 and POL4 more than POL3. In conclusion, POL3 and POL4 have differential functions in NHEJ, independent of the RAD50-mediated repair pathway

Genome-Wide High-Resolution Mapping of UV-Induced Mitotic Recombination Events in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae and most other eukaryotes, mitotic recombination is important for the repair of double-stranded DNA breaks (DSBs). Mitotic recombination between homologous chromosomes can result in loss of heterozygosity (LOH). In this study, LOH events induced by ultraviolet (UV) light are mapped throughout the genome to a resolution of about 1 kb using single-nucleotide polymorphism (SNP) microarrays. UV doses that have little effect on the viability of diploid cells stimulate crossovers more than 1000-fold in wild-type cells. In addition, UV stimulates recombination in G1-synchronized cells about 10-fold more efficiently than in G2-synchronized cells. Importantly, at high doses of UV, most conversion events reflect the repair of two sister chromatids that are broken at approximately the same position whereas at low doses, most conversion events reflect the repair of a single broken chromatid. Genome-wide mapping of about 380 unselected crossovers, break-induced replication (BIR) events, and gene conversions shows that UV-induced recombination events occur throughout the genome without pronounced hotspots, although the ribosomal RNA gene cluster has a significantly lower frequency of crossovers