title: Pol3 is involved in nonhomologous end-joining in Saccharomyces cerevisiae.
abstract: Nonhomologous end joining connects DNA ends in the absence of extended sequence homology and requires removal of mismatched DNA ends and gap-filling synthesis prior to a religation step. Pol4 within the Pol X family is the only polymerase known to be involved in end processing during nonhomologous end joining in yeast. The Saccharomyces cerevisiae POL3/CDC2 gene encodes polymerase delta that is involved in DNA replication and other DNA repair processes. Here, we show that POL3 is involved in nonhomologous end joining using a plasmid-based end-joining assay in yeast, in which the pol3-t mutation caused a 1.9- to 3.2-fold decrease in the end-joining efficiency of partially compatible 5' or 3' ends, or incompatible ends, similar to the pol4 mutant. The pol3-t pol4 double mutation showed a synergistic decrease in the efficiency of NHEJ with partially compatible 5' ends or incompatible ends. Sequence analysis of the rejoined junctions recovered from the wild-type cells and mutants indicated that POL3 is required for gap filling at 3' overhangs, but not 5' overhangs during POL4-independent nonhomologous end joining. We also show that either Pol3 or Pol4 is required for simple religation of compatible or blunt ends. These results suggest that Pol3 has a generalized function in end joining in addition to its role in gap filling at 3' overhangs to enhance the overall efficiency of nonhomologous end joining. Moreover, the decreased end-joining efficiency seen in the pol3-t mutant was not due to S-phase arrest associated with the mutant. Taken together, our genetic evidence supports a novel role of Pol3 in nonhomologous end joining that facilitates gap filling at 3' overhangs in the absence of Pol4 to maintain genomic integrity.
authors.affiliation: Department of Pathology and Radiation Oncology, David Geffen School of Medicine at UCLA, University of California-Los Angeles, 650 Charles E. Young Drive South, Los Angeles, CA 90095, USA.
authors.forename: Cecilia Y
authors.initials: CY
authors.lastname: Chan
authors.forename: Alvaro
authors.initials: A
authors.lastname: Galli
authors.forename: Robert H
authors.initials: RH
authors.lastname: Schiestl
mesh_terms: D001483:Base Sequence; D043171:Chromosomal Instability; D004249:DNA Damage; D004258:DNA Polymerase III / Q000502:physiology*; D004260:DNA Repair; D004259:DNA-Directed DNA Polymerase / Q000502:physiology; D009154:Mutation; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000502:physiology*; D029702:Schizosaccharomyces pombe Proteins / Q000502:physiology
chemical_list: C491232:POL3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; C503462:POL4 protein, S pombe; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase