A genome wide screen to identify genes that prevent the accumulation of mutations

Synthesis of eukaryotic genomes requires the function of DNA replicative polymerases Polα (Pol1), Polɛ (Pol2) and Polδ (Pol3). Biochemical evidence supports the idea that Polα initiates DNA synthesis at replication origins as well as at every Okazaki fragment. This initial DNA synthesis is further extended by the proofreading proficient DNA polymerases Polɛ and Polδ. The DNA lagging strand is synthesized by Polδ in a discontinuous manner, as 200–300 bases‐long Okazaki fragments. On the other hand, the leading strand is synthesized in a continuous manner; however, it is less clear if Polɛ, Polδ or a combination of both, are mainly required during this process. In order to identify unrecognized genes that might affect DNA replication fidelity, we performed a genome‐wide screen in S. cerevisiae searching for synergistic mutator interactions with DNA polymerase mutants. We modified the Synthetic Genetic Array protocol to cross low‐fidelity DNA polymerase mutants pol1‐L868M , pol2‐M644G and pol3‐L612M (or wild‐type) against the non‐essential yeast gene deletion collection. Single and double mutants were tested for an increased mutator phenotype with in vivo mutator assays ( lys2‐10A and CAN1 inactivation). Analysis of the single mutants identified ~45 genes, with well‐known functions in different DNA repair pathways, which prevented the accumulation of mutations. Moreover, we identified one additional gene deletion that has not been previously linked to a mutator phenotype. This mutant not only resulted in elevated mutation rates, but also increased gross chromosomal rearrangements. Analysis of the double mutants identified a small number of gene deletions (including exo1Δ ) that resulted in a strong synergistic mutator interaction with pol1‐L868M and pol3‐L612M , but not with pol2‐M644G. This unpredicted bias supports the hypothesis that leading and lagging strand are mainly synthesized by Polδ. Furthermore, we have identified a small number of genes whose roles become increasingly important under conditions of impaired DNA replication fidelity. Support or Funding Information Deutsches Krebsforschungszentrum (DKFZ). Marie Curie CIG grant “iMMR”.