Rqh1 blocks recombination between sister chromatids during double strand break repair, independent of its helicase activity

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.

Fig. 1.

A schematic depiction of the Th805 system and the predicted products that can form after repair of a DSB induced at MATa.

Fig. 2.

Bar graphs showing comparisons in GC and SCC/NHEJ frequencies in various genetic backgrounds. Each bar represents an average of a minimum of three individual experiments. (a) Comparison of SCC/NHEJ and GC rates among Th805 (wild type), Th805 Δrqh1 (rqh1), Th805 Δpku80 (pku80), and Th805 Δrqh1 Δpku80 (rqh1 pku80). (b) Comparison of Th805 wee1-50 (wee1-50), Th805 Δrqh1 wee1-50 (rqh1 wee1-50), Th805 Δpku80 wee1-50 (rqh1 pku80), and Th805 Δrqh1 Δpku80 wee1-50 (rqh1 pku80 wee1-50). (c) Comparison of Th805 (wild type), Th805 Δrqh1 (rqh1), and Th805 rqh1-K547I (rqh1 K547I).

Fig. 3.

Demonstration of cleavage by HO endonuclease rad22-YFP was introduced into Th805 in a wild-type background. HO endonuclease was induced by growth in media lacking thiamine and the cells followed for 30 h. Before induction, only a few cells ≈2–4% showed Rad22-YFP foci. By ≈16 h postinduction, foci were visible in many more cells (≈30%). By 28 h, nearly all cells (>77%) contained Rad22-YFP foci, suggesting that essentially every cell had received at least one HO-induced break by this time.

Fig. 4.

HO cleavage is equivalent in wild-type and Δrqh1 backgrounds. Cultures of Th805 and Th805 Δrqh1 were grown for 20 or 24 h in media lacking thiamine to induce the HO endonuclease to cleave at the MATa locus. EcoRI-digested genomic DNA was isolated and used for Southern blot analysis. The blot was probed with a ³²P-labeled kanMX6 probe (black bar shown in diagram). The autoradiograph shows that equivalent levels of the 3.5-kb band generated by a combination of EcoRI and HO endonuclease cutting are seen at equivalent levels in both wild-type (WT) and Δrqh1 backgrounds.