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 ⌬rqh1 cells, repair by SCC is favored, and gene conversion rates slow significantly. When we limited the potential for SCC in ⌬rqh1 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.homologous recombination ͉ nonhomologous end joining ͉ rqh1 ͉ gene conversion D ouble strand breaks (DSBs) pose a major problem for genomic instability and cell survival, because a single unrepaired DSB is, presumably, sufficient to cause cell death (1). The sources of DSBs can be either endogenous, such as those induced during the reshuffling of DNA in Ig gene diversification, or exogenous, such as those induced by exposure to ionizing radiation (2-4). The cell has two major mechanisms for the repair of DSBs: homologous recombination (HR) and nonhomologous end joining (NHEJ), each used to varying degrees in different organisms (2,3,5,6). HR is characterized as an error-free process using homologous sequences as the template to repair the DSB. Repair by HR begins with the formation of 3Ј single strand ends at the break that can then invade homologous duplex DNA. The 3Ј end of the invading strand is extended by DNA polymerase. At this point, the DSB can be repaired by either DSB repair, which involves formation of a double Holliday junction (HJ) or synthesis-dependent strand annealing (3, 7).In NHEJ, the DNA ends are resealed by rejoining the broken ends; however, this process can lead to loss of information at the break and is, thus, referred to as error-prone repair (5,8). Whereas mammalian cells preferentially use NHEJ over HR, budding yeast, for the most part, use HR over NHEJ. In Saccharomyces cerevisiae, the MRX (MRN in Schizosaccharomyces pombe) complex has been shown to play a role in NHEJ (9, 10). In S. pombe, only pKu70͞80 and Ligase IV have been identified as functioning in NHEJ, and MRN does not appear to be active in this process (11).RecQ DNA helicases are found in vi...