recombination have been revealed in the absence of Mus81. While yen1D mutants are repair proficient, mus81D yen1D double mutants exhibit a higher sensitivity to DNA-damaging agents that disturb replication fork progression than mus81D mutants [28,30,31]. Together, these data point out that Mus81 and Yen1 may promote a replication fork restart mechanism. In vitro, Yen1 is a specialized Holliday junction resolvase [26] whereas the Mus81-Mms4 complex prefers branched DNA substrates that contain a discon- tinuity or a nick adjacent to the branch point, but also cleaves normal HJs [27,59–61]. Here, we have demonstrated genetically that both Mus81 and Yen1 were required for efficient BIR. According to previously published data, we propose that Mus81 and Yen1 would act to establish the replication fork required for BIR by processing recombination intermediates such as D-loops or HJs. Nevertheless, BIR still occurred at a low frequency in mus81D yen1D mutants, suggesting that other factors could promote this critical step of BIR in the absence of Mus81 and Yen1. Interplay between Slx4, Mus81, and Yen1 causes template switching and half-crossovers Our results are consistent with additional roles of Mus81 and Yen1 in later steps of BIR. We have demonstrated that Mus81, Slx4 and Yen1 were required together for efficient template switching during BIR. The mus81D slx4D yen1D mutants showed an increased occurrence of T7/3-MAT translocations (Figure 5B), which we infer as being partly due to a defect in template switching from MAT to the HM loci (Figure 6). However, we did not observe any increase of T7/3-MAT in mus81D slx1D yen1D mutants, even though Slx1 is the catalytic subunit of Slx1-Slx4 nuclease heterodimer. On the contrary, we observed a WT or decreased level of T7/3-MAT translocations in all slx1D mutants. We concluded that Slx4 and Slx1 act independently in BIR, presumably because of Slx4 additional functions apart from Slx1 at the replication fork [51,62,63]. Regarding the involvement of SSEs in half-crossover production, the absence of Mus81 or Slx1 significantly decreased the amount of circular chromosomes III among translocants (Figure 7C). Notably, no further decrease was observed when removing Mus81, Slx1/Slx4 or Yen1, all of which have been involved in crossover formation during meiosis in yeast [64,65]. This could be due to the involvement of other nucleases such as Mlh3 and Exo1, as recently reported during the revision of this manuscript [65]. Our assay does not permit a direct analysis of the role of SSEs in half-crossover since the formation of circular chromosomes III is limited by the frequencies of translocations, template switching and HMR cleavage. In principle, HML could also be cleaved upon invasion so that an HMR/HML double cleavage could lead to a circular chromosome III by an SSA-like mechanism. However, this hypothesis is not supported by our results as we observed a similar frequency of circular chromosomes III in rad1D mutants and WT. Instead, circularization of chromosome III via SSA would generate a heterologous single-stranded DNA overhang that would require Rad1 for its removal [66] and, indeed, we have observed a requirement of Rad1 in the formation of T7/3-HMR translocations via BIR/SSA in our assay (Figure 5 and Figure 6). Despite the limitations of our assay, our genetic data suggest an interesting interaction between Mus81, Yen1 and Slx4 SSEs. Whereas mus81D translocants showed a decrease in the frequency of circular chromosomes III, mus81D slx4D translocants showed a significant increase, which was suppressed in the additional absence of Yen1 (Figure 7C). These results suggest that Slx4 may have a specific role in regulating the ability of Mus81 and Yen1 to catalyze half-crossovers. It has been previously shown that Mus81 is involved in half-crossovers following BIR [23] and that Nuclease Processing of BIR Intermediates Mus81 and Yen1 independently promote crossovers during gene conversion, Yen1 serving as a backup function in mus81D cells [30]. However, here we uncover two parallel pathways, one using Mus81 and Slx4 and the other Yen1. This is in agreement with a similar involvement recently described for these nucleases in two pathways of crossover formation during sister-chromatid recom- bination [67]. It remains unclear how Slx4 may regulate Mus81 and Yen1. A recent cell-cycle analysis of Mus81-Mms4 and Yen1 revealed that their catalytic activities are regulated by phosphor- ylation events. In mitotic cells, Mus81-Mms4 is hyperactivated by Cdc5-mediated phosphorylation at G2/M while Yen1 is activated later by dephosphorylation in M phase [27]. Nevertheless, it remains unknown if Yen1 can be activated earlier in the absence of Mus81 or upon DSB induction. Mec1/Tel1 kinases phosphor- ylate Slx4 in response to DNA damage [50,51] and may participate in modulating context-specific protein interactions between Slx4, Mus81 and Yen1 and allow substrate accessibility to activated Mus81 and Yen1. Altogether, our results suggest that Slx4 plays a central role during BIR. Slx4 may regulate Mus81 and Yen1, whose cleavage activities are required for replication fork establishment and could either cause template switching or half-crossovers. In the case of one-ended DSBs, it has been proposed that dynamic displacement of the invading strand out of the D-loop would contribute to template switching [16]. This implies that the invading strand would be displaced early during BIR, after a short tract of DNA synthesis. Nevertheless, events of template switching have been observed in later steps of BIR, as far as 10-kb downstream of the site of invasion [16,23]. Such a synthesis would expose long tracts of single-stranded DNA if it were the result of the sole extension of the invading strand. Despite the fact that such long single-stranded DNA tails have been involved in gene conversion events monitored in mitotic gap repair assays, we propose that, at some point, priming of lagging strand synthesis would ensure a better protection of the recombination interme- diates, safeguarding genome stability. Thus, we propose that template switching events would happen after the establishment of the BIR fork and priming of lagging strand synthesis. In vitro data showed that canonical replication forks are among the preferred substrates of Mus81-Mms4 and Yen1 [25,68], therefore we propose that Mus81, Slx4 and Yen1 would act on the replication fork during BIR to cause template switching and half-crossovers. An integrated model to explain BIR–mediated chromosomal rearrangements and the role of structure- selective endonucleases Our results together with previous data permit us to propose a new model for BIR and the role of the different SSEs used in this study (Figure 8). During HR, priming of synthesis from the 39 invading end extends the initial D-loop and failure to capture the other DSB end would promote BIR. We propose that Mus81 would cleave the extended D-loop structure to allow the establishment of a replication fork. In the absence of Mus81, branch migration of the D-loop would create an intact Holliday junction, which could be processed by Yen1 with the same outcome. Pol32 would promote extensive DNA synthesis and complete replication would generate a non-reciprocal translocation (NRT). We propose that the BIR fork could stall and be processed by Mus81-Slx4-Yen1 to cause template switching ([a], Figure 8). Differential cleavage of the BIR fork by Mus81-Slx4-Yen1 would terminate BIR at the expense of a half- crossover ([b], Figure 8). Altogether, this work brings a clearer view about the involvement of SSEs in the BIR mechanism of DSB repair. Importantly, we show that SSEs are involved in replication PLOS Genetics | www.plosgenetics.org 14 September 2012 | Volume 8 | Issue 9 | e1002979