Abstract
Many questions remain about the process of DNA double strand break (DSB) repair by homologous recombination (HR), particularly concerning the exact function played by individual proteins and the details of specific steps in this process. Some recent studies have shown that RecQ DNA helicases have a function in HR. We studied the role of the RecQ helicase Rqh1 with HR proteins in the repair of a DSB created at a unique site within the Schizosaccharomyces pombe genome. We found that DSBs in rqh1(+) cells, are predominantly repaired by interchromosomal gene conversion, with HR between sister chromatids [sister-chromatid conversion (SCC)], occurring less frequently. In Deltarqh1 cells, repair by SCC is favored, and gene conversion rates slow significantly. When we limited the potential for SCC in Deltarqh1 cells by reducing the length of the G2 phase of the cell cycle, DSB repair continued to be predominated by SCC, whereas it was essentially eliminated in wild-type cells. These data indicate that Rqh1 acts to regulate DSB repair by blocking SCC. Interestingly, we found that this role for Rqh1 is independent of its helicase activity. In the course of these studies, we also found nonhomologous end joining to be largely faithful in S. pombe, contrary to current belief. These findings provide insight into the regulation of DSB repair by RecQ helicases.
Highlights
D ouble strand breaks (DSBs) pose a major problem for genomic instability and cell survival, because a single unrepaired double strand break (DSB) is, presumably, sufficient to cause cell death [1]
In wild-type cells, DSBs are repaired preferentially through gene conversion (GC) and that this choice is regulated by the action of Rqh1 by suppressing sisterchromatid conversion (SCC) leading to increased GC
Colonies were analyzed for chromosome loss (ChL), GC, SCC, or nonhomologous end joining (NHEJ), as described in Methods and in supporting information, which is published on the PNAS
Summary
Repair of Site-Specific DSB in the Wild-Type Background. A unique system was used to analyze the repair of a site-specific DSB at Conflict of interest statement: No conflicts declared. An ectopic MATa site through the expression of the HO endonuclease, under control of the thiamine-repressible promoter nmt (Fig. 1) [35]. Strains carrying the minichromosome with the MATa site are referred to as TH805. Colonies were analyzed for chromosome loss (ChL), GC, SCC, or NHEJ, as described in Methods and in supporting information, which is published on the PNAS web site. In wild-type cells after 48 h of induction, Ϯ 5% of colonies remained Adeϩ but became G418-sensitive, indicative of repair by GC, whereas Ϯ 4% of colonies remained both Adeϩ and G418R, consistent with repair by SCC or NHEJ. At 72 h postinduction, 60 Ϯ 7% of colonies had repaired the DSB by GC, whereas 17 Ϯ 7% remained Adeϩ G418R
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