little or no effect on the frequency of deletion-types, although the low-level of conversion-types is abolished (Figure 3B and D). This indicates that deletion-types induced by the lacO-LacI RFB occur mainly by a mech- anism other than Rad51-mediated strand invasion. One possibility is SSA, which in Saccharomyces cerevisiae is known to be dependent on Rad52 and the Rad1–Rad10 structure-speciﬁc endonuclease (33). We therefore assessed lacO-LacI-induced recombinant formation in rad22D (Rad22 is the S. pombe orthologue of Rad52) and swi10D (Swi10 is the S. pombe orthologue of Rad10) single and double mutant strains (Figure 3A and C and Supplementary Table S1). Deletion-types are reduced in both single mutants and almost abolished in the double mutant. This loss of recombinant formation is not due to ﬂuctuations in the levels of LacI expression, which, based on western blot analysis, remain fairly constant in the strains under investigation here (Supplementary Figure S1). Therefore these data indicate that lacO- LacI-induced deletion-types are likely to be formed by SSA. The ﬁnding that lacO-LacI-induced deletion-types are formed by SSA suggests that replication fork stalling at the lacO-LacI RFB can result in DSBs. Indeed an HO-induced DSB between the ade6 repeats has previous- ly been shown to give rise to deletion-types via SSA (24). For such breaks to be repaired by SSA between the ade6 repeats, several kilobases of DNA would have to be resected on either side of the DSB. Extensive 50–30 DSB resection requires either of two alternative pathways—one dependent on the exonuclease Exo1 and the other on a RecQ family DNA helicase (Sgs1 in S. cerevisiae or BLM in humans) (34). To determine whether lacO-LacI-induced deletion-types depend on DNA resection by Rqh1 (= S. pombe RecQ helicase) and/or Exo1 we assessed recombin- ant formation in rqh1D and exo1D single and double mutant strains (Figure 3A and C and Supplementary Table S1). Loss of Rqh1 reduces lacO-LacI(+)-induced deletion-type formation by 2-fold (P < 0.00001), whereas recombinant formation remains largely unaffect- ed in the exo1D single mutant (P = 0.17) (Figure 3A). Importantly the rqh1D exo1D double mutant exhibits a synergistic reduction ( 10-fold) in deletion-types (P < 0.00001) (Figure 3A). With LacI (++) the effect of rqh1 and exo1 deletion is less pronounced with only a slight reduction in recombinants in each single mutant and an almost 2-fold reduction in the double mutant (P < 0.0001). We suspect that this less dramatic reduction in recombinants with LacI (++) is due to the greater fre- quency of DNA breakage, which can occur in successive cell cycles if repair is mediated by annealing between the lacO repeats without deleting the entire array. This means that there are multiple opportunities of forming an Ade+ recombinant, and therefore mutants that are not com- pletely deﬁcient in SSA will accumulate Ade+ recombin- ants to a similar level as wild-type albeit at a slower rate. Consistent with this there is a 5-fold difference in Ade+ recombinant frequency between wild-type and rqh1D exo1D strains after 24 h of LacI (++) induction in liquid culture (Figure 4F) as opposed to only a 2-fold difference after 6 days induction on solid media (Figure 3C). Nucleic Acids Research, 2011, Vol. 39, No. 15 6575 Overall the data indicate that Rqh1 or Exo1 are needed for efﬁcient SSA, presumably because they promote DNA resection. However, RecQ helicases and Exo1 are also implicated in the processing of stalled replication forks and therefore we cannot discount the possibility that they affect recombinant formation by other mechanisms (35–37). lacO-LacI promotes DSB formation A requirement for SSA is the presence of two DNA ends, which are resected to expose complementary nucleotide sequences. CHEF gel and Southern blot analysis of chromosome 3 revealed a band that migrates ahead of the chromosome 3 band, indicative of a DSB and amounting to 3% of the total DNA, in samples from cells with the lacO array after 20 h of LacI (++) induction (Figure 4A and B, compare lanes a and b, and i and j). Moreover, the detection of this band using probes either side of ade6 indicate that the chromosome is broken into two fragments. If the band is indicative of broken DNA, which is subject to active repair, then it should accumulate to higher levels in rqh1D exo1D and rad50S mutants that are defective in processing DSBs. In both mutants, but especially in the rqh1D exo1D double mutant, the DSB band accumulated to a higher levels (16.8 and 6.1%, re- spectively) than in the wild-type consistent with the expected impairment in DSB repair (Figure 4B, lanes c–f and k–n). However, it should be noted that RecQ helicases and Exo1 have roles in promoting the stability of stalled forks (35–37) and therefore the accumulation of high levels of DSBs in the rqh1D exo1D double mutant could also be due to increased levels of DNA breakage. To conﬁrm that the detected band is indeed the result of a DSB at ade6, we analysed samples from a strain contain- ing the HO endonuclease cleavage site MATa inserted in place of the lacO array (Figure 4B, lanes g, h, o and p). Following induction of the HO endonuclease a DSB band was detected, which migrated similarly to the band detected in the samples from the lacO-LacI(++)-contain- ing strains (lanes h and p). Finally, we analysed DNA after digestion with AvrII, which liberates the ade6 region as a 58.3 kb fragment (Figure 4C). In addition to the full-length band, a 24 kb band was detected in sam- ples where LacI (++) was induced, which maps to within the lacO array (Figure 4D, lanes b and d). The amount of DNA breakage is much higher than in Figure 4B with 20% of the DNA broken in the wild-type and 84% in the rqh1D exo1D double mutant. This is due to a longer induction time for LacI (++) (24 h instead of 20 h), which ensures that all cells have undergone at least one cell div- ision in the presence of its maximum level (see earlier). Altogether these data suggest that replication fork stalling at the lacO-LacI RFB causes DNA breakage in a signiﬁ- cant percentage of cells. The lacO-LacI array reduces the viability of mutants defective in SSA The high levels of DSBs detected in strains containing the lacO array upon LacI (++) induction should correlate with a reduction in viability in mutants deﬁcient in DSB