Unprocessed flaps of Okazaki fragments are repaired by homologous recombination initiated by double-strand breaks

Abstract

Saccharomyces cerevisiae Dna2, which possesses endonuclease and helicase activities, plays a critical role in Okazaki fragment processing. To gain further insight into other genes acting in conjunction with Dna2 during Okazaki fragment processing, we performed systematic synthetically lethal (or sick) screening using mutant cells containing dna2Δ405N, a dna2 allele lacking the N-terminal 405 amino acids that displays a temperature-sensitive growth defect. This approach aims to define the pathways that are critical to repair DNA lesions caused by dysfunction of the dna2 mutation. Moreover, it could allow us to find a bypass pathway, in which cells do not require a functional Dna2 for processing of Okazaki fragments. We found that dna2Δ405N exhibits synthetic growth defect with several gene involved in DNA repair such as srs2Δ and sgs1Δ, which plays a role in double-strand -break (DSB)-initiated homologous recombination repair. In support of this, rad51Δ, a yeast paralog of RecA, showed growth defect with dna2Δ405N when DNA damages were imposed. As expected, a mutation in TOP3 encoding topoisomerase III, is also synthetic lethal with dna2Δ405N, since TOP3 is required for a normal function of SGS1. In addition, we found that defects in POL32 and PIF1, the two genes implicated in displacement DNA synthesis that lead to flap formation, suppressed temperature-sensitive phenotype of dna2Δ405N. Moreover, pif1Δ suppressed the null mutation of dna2. These findings suggest either that the length of flap in the absence of pif1 or pol32 mutation is extremely short or that flaps do not form altogether, thus allowing the cells to bypass the requirement of a functional Dna2. In summary, our results demonstrate that replication errors accumulated by dysfunction of Dna2 during Okazaki fragment processing are corrected largely by DSB-initiated homologous recombination and that the requirement of Dna2 can be bypassed by suppressing the displacement DNA synthesis, most li...