Nuclease Processing of BIR Intermediates Figure 2. Template switching occurs between MATa-inc, HMR, and HML loci. (A) Schematic representation of chromosomes VII and III showing the positions of the PCR primers, EcoRV sites (EV) and the 7L probe used for the analysis. Distances between the primers and EcoRV sites and the HO cut sites are indicated. (B) Appearance of BIR repair product, as monitored by PCR, in the WT strain. DNA samples used for PCR were extracted at intervals after HO induction with galactose. PCR reactions were performed with the p7 primer and either the p3-M, p3-R or p3-L primer (see panel A). A primer pair corresponding to ACT1 locus on chromosome VI was used to control the amount of genomic DNA used for PCR at each time point. 35 cycles of PCR amplifications were performed. (C) Appearance of BIR repair products, as monitored by Southern analysis with the 7L probe, in the WT strain. DNA samples were extracted as for the PCR assay. Positions of the bands corresponding to 7/3-MAT, 7/3-HMR and 7/3-HML intermediates, and to the uncut and cut chromosome VII (chr. VII) are indicated. Two genomic DNA samples coming from survivor colonies containing T7/3-MAT and T7/3-HMR translocations were included in the experiment. GAL, galactose; h, hours. (D) Schematic representation of BIR events involving template switching likely occurring in our assay. Black arrows indicate the first invasion events while red arrows indicate the secondary template switching events. The different possible final outcomes are depicted. The red cross indicates the presence of inc non-cleavable sequences. M; MAT; R, HMR; L, HML. See text for more details. doi:10.1371/journal.pgen.1002979.g002 for BIR [11,14]. Using translocants containing T7/3-MAT and T7/3-HMR translocations, we confirmed the size of the repair intermediates detected by Southern (Figure 2C, right). We did not recover any translocant containing translocations corresponding to repair intermediates detected at HML. Together, these data show that one unique feature of our experimental system is that it allows the detection of events of template switching between MATa-inc and HML and possibly between MATa-inc and HMR during chromosomal rearrange- ment. These events of template switching likely participated to give rise to the formation of T7/3-MAT and T7/3-HMR translocations and dicentric chromosomes resulting from BIR completed from HML (Figure 2D). Because dicentric chromosomes are known to be unstable [49], this may be why we could not detect such type of chromosomal rearrangement. We noted that BIR initiation at HMR by the broken chromosome VII would restore an HO cleavable site that might not be properly silenced in the resulting translocations. In the latter case, HO cleavage would destabilize these translocations and their stabilization would require repair by gene conversion (GC) using the non-cleavable sequences at MATa-inc. Mus81 and Yen1 promote chromosomal rearrangements Observed chromosomal rearrangements likely occurred by BIR through the invasion of the MATa-inc and HMR loci. Homology of the centromeric 198-bp Ya-Z1 fragment present at one DSB end would be used to invade MATa-inc or HMR loci at the Ya region and to duplicate chromosome III sequences until reaching its right telomere, generating T7/3-MAT and T7/3-HMR NRTs (type a, Figure 3). Alternatively, since we observed the cleavage of chromosome III as a consequence of HMR invasion, it is also possible that T7/3-HMR translocations occurred by BIR followed by single-strand annealing (SSA), co-segregating with an intact chromosome III (type b, Figure 3). Finally, cleaved chromosome III could circularize and co-segregate with the translocation (type c, Figure 3). To confirm genetically the involvement of BIR in the generation of translocations using our experimental system, we assayed mutants of Rad52, Rad51 and Pol32 as representative key functions in BIR. Next, we asked whether DNA nucleases acting on branched structures such as D-loops, replication forks or HJs would have a role in the cascade of recombination events that led to translocations. We chose to study the genetic role of Mus81, Rad1, Yen1, Slx1 and Slx4 because of their known functions in recombination processes [25]. Because none of the genes coding for these SSEs are essential for viability, we assayed deletion mutants and combined mutants between them to assay redun- dancy of functions. Although Slx1 and Slx4 form a heterodimer complex, such as Mus81-Mms4 and Rad1–Rad10, we decided to study slx1D and slx4D mutations separately because Slx4 seems to have additional roles in DSB repair apart from regulating the Slx1 nuclease activity [50,51]. We also included the DNA helicase Sgs1 because of its known role in HJ dissolution (Figure 4A). For each mutant assayed, we recovered survivors after HO endonuclease induction and selected colonies that contained translocations between chromosome VII and chromosome III, as described above (5-FOA-resistant colonies). We did not recover any translocants in rad52D and rad51D mutants, in which strand invasion fails to occur (Figure 4A). POL32 deletion reduced 4-fold the frequency of translocations in comparison with the WT (x2, p,0.01), arguing in favor of the involvement of the Pol32- dependent BIR pathway in the generation of the translocations analyzed in this study. We did not observe any increase of translocations in sgs1D cells, even though SGS1 gene had been identified as a suppressor of translocations involving template switching events [19]. Among the nuclease single mutants, only mus81D showed a slight but significant decrease in the frequency of translocants (x2, p,0.01) when compared to the WT. In contrast, we observed significant decreases in the frequency of translocants between the WT, mus81D rad1D and mus81D rad1D slx1D mutants, but these effects were found to be epistatic with mus81D (Figure 4A; 2 x , p,0.01). On the contrary, we observed further significant decreases (x2, p,0.01) in mus81D yen1D mutants, of about 3.5-fold and 2.5-fold compared to the WT and mus81D, respectively (Figure 4A). We conclude that Mus81 and Yen1 are both required for translocations in our assay. Since it existed the possibility that the translocations were also produced by BIR/SSA, we assayed translocation formation in an HMR-inc strain, in which HMR locus would not be susceptible to HO cleavage and translocations would only be produced by BIR. We observed that survival dropped from 82% in the original HMR WT strain to 52% in the HMR-inc WT strain, although the frequency of translocants remained around 40% (Figure 4B and Figure S1). In the control HMR-inc background, POL32 deletion reduced 18.5-fold the frequency of translocations in comparison with the WT (Figure 4B; x2, p,0.01). This result demonstrates a clear dependency of translocations on the Pol32-dependent BIR pathway in this background. We observed a significant decrease of 3.4-fold in HMR-inc mus81D yen1D mutants compared to the HMR-inc WT but no decrease in the HMR-inc mus81D and HMR- inc yen1D single mutants (Figure 3B; x2, p,0.01). These results indicate that Mus81 and Yen1 have overlapping functions in BIR. To confirm this, we performed time-course experiments to monitor the kinetics of appearance of BIR intermediates in these mutants (Figure 4C). Kinetics of DSB formation in the mutants was similar to the WT, allowing us to directly compare the accumulation of BIR intermediates at each time point (Figure 4D). In HMR-inc WT cells, 7/3-HMR and 7/3-HML intermediates appeared 4 h and 5 h after DSB induction, respectively (Figure 4C, 4D). 7/3-MAT intermediates were detected 6 h after DSB induction (Figure 4C, 4D), showing that their delayed appearance in the original HMR WT strain was partly due to HMR cleavage. PLOS Genetics | www.plosgenetics.org 6 September 2012 | Volume 8 | Issue 9 | e1002979