The DNA polymerase delta (POL3/CDC2) allele pol3-t of Saccharomyces cerevisiae has previously been shown to be sensitive to methylmethanesulfonate (MMS) and has been proposed to be involved in base excision repair. Our results, however, show that the pol3-t mutation is synergistic for MMS sensitivity with MAG1, a known base excision repair gene, but it is epistatic with rad50Delta, suggesting that POL3 may be involved not only in base excision repair but also in a RAD50 dependent function. We further studied the interaction of pol3-t with rad50Delta by examining their effect on spontaneous, MMS-, UV-, and ionizing radiation-induced intrachromosomal recombination. We found that rad50Delta completely abolishes the elevated spontaneous frequency of intrachromosomal recombination in the pol3-t mutant and significantly decreases UV- and MMS-induced recombination in both POL3 and pol3-t strains. Interestingly, rad50Delta had no effect on gamma-ray-induced recombination in both backgrounds between 0 and 50 Gy. Finally, the deletion of RAD50 had no effect on the elevated frequency of homologous integration conferred by the pol3-t mutation. RAD50 is possibly involved in resolution of replication forks that are stalled by mutagen-induced external DNA damage, or internal DNA damage produced by growing the pol3-t mutant at the restrictive temperature.

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.