Nuclease Processing of BIR Intermediates Figure 7. PFGE analysis of chromosome III circularization in SSE mutants. (A) Schematic representation of circularization of chromosome III by BIR plus half-crossover. Arrows indicate different ways of intermediate cleavage. (B) PFGE followed by Southern analysis using the 3R2 probe (See Figure 1G) of Ura2 5-FOAr survivors in WT, pol32D, mus81D, slx4D, mus81D slx4D and mus81D slx4D yen1D strains. Black arrows indicate the positions of T7/3-MAT translocations, and the circular and linear chromosomes III (Chr. III). PFGE, pulse-field gel electrophoresis. (C) Graphical plotting of percent of translocants containing circular chromosome III for each strain tested. (D) Frequency of chromosome III circularization among translocants. ** and *, differences with the WT statistically significant (p,0.01 and p,0.05, respectively, x2 with Yates’ correction) 6, differences with mus81D slx4D statistically significant (p,0.01, x2 with Yates’ correction). Error bars represent standard deviations. See Table S1 for complete statistical analysis. doi:10.1371/journal.pgen.1002979.g007 DSB. DSB repair occurred by recombination between repeated DNA sequences dispersed over two different chromosomes and generated translocations and circularization of chromosomes at a high frequency. Our data indicate that chromosomal rearrange- ments occurred primarily by BIR, a HR sub-pathway that involves the invasion of an intact homologous DNA duplex by only one end of the DSB and subsequent replication primed from the invading strand. We have shown with this assay that the structure-selective endonuclease (SSE) factors Mus81, Yen1, and Slx4 may process recombination intermediates at different steps during BIR and cause template switching and half-crossovers. In our assay, induction of the HO endonuclease produced a DSB on chromosome VII. Translocants can only be detected if both chromatids were broken in G2 phase (Figure 3), otherwise the DSB would be preferentially repaired by sister-chromatid recombination [53,54], restoring the HO cut site. DSB ends on chromosome VII display homology to MAT and HMR loci that is mainly restricted to one end (198-bp out of 240-bp). As previously described, this feature favors DSB repair by BIR against a conventional gene conversion mechanism [11]. The centromeric 198-bp DSB end would invade MAT or HMR at the Ya region and prime BIR synthesis to produce the observed translocations, which contained chromosome III sequences from MAT or HMR to the telomere fused to chromosome VII at the break site, termed T7/3-MAT and T7/3-HMR translocations. Consequently, the loss of the chromosome VII arm, distal to the DSB and containing the URA3 marker and non-essential genes, would lead to the 5-FOA-resistant (Ura2) phenotype of the translocants (type a, Figure 3). Eventually, the URA3 marker could be used to convert the endogenous ura3-1 allele on chromosome V and lead to the rarely observed Ura+ cells containing translocations (Figure S2B). Alternatively, secondary cleavage of HMR upon its invasion would promote repair by BIR/SSA and the production of the same types of translocations (type b, Figure 3). HMR cleavage would stimulate the circularization of chromosome III, which could co-segregate with the translocation (type c, Figure 3). Repair by BIR/ SSA and chromosome III circularization do not happen in the HMR- inc background. The latter observation discards the possibility that the T7/3 translocations were the products of half-crossovers, with the translocation co-segregating with the uninvolved chromatid of chromosome III (analogous to type b, Figure 3), since breakage of chromosome III would stimulate its circularization. Lastly, DSB repair by GC with inc non-cleavable sequences or by erroneous NHEJ could seal the break leading to a mutated and non-cleavable form of HO site without chromosomal rearrangement and would maintain the URA3 marker on chromosome VII. This would lead to the formation of Ura+ survivors. (type d, Figure 3). Altogether, the differential repair of both DSBs on chromosome VII chromatids gave rise to the observed Ura+/Ura2 phenotypes of survivor colonies. Absence of Pol32 impedes template switching and forces crossover formation An important aspect that remains unclear is the apparent instability of the BIR fork, which may be cleaved during its advance, promoting template switching or producing half-cross- overs. All essential DNA replication factors except those for pre- replication complex assembly are required for BIR [14], playing in favor of fork stability. However, the DNA damage checkpoint is activated during BIR and dNTP levels are elevated to facilitate repair, which is thought to happen in the G2 phase of the cell cycle [11,13,55]. Consequently, DNA synthesis during BIR has been found to be highly inaccurate [56] and replication fork progression may be perturbed by the absence of S phase-specific factors. Interestingly, the nonessential DNA polymerase d subunit Pol32 seems to represent a key factor for BIR completion but performs a function that is still unknown. In our assay, we observed that pol32D mutants had a clear defect in producing translocations, but not as strong as the one observed in rad52D and rad51D mutants (Figure 4A), confirming that some translocations were produced via BIR/SSA, in which extensive DNA synthesis would not be required. However, Pol32 became essential for translocations in the absence of HMR cleavage in the control HMR-inc strains (Figure 4B). In these strains, we could not detect any BIR intermediate (Figure 4C), meaning that Pol32 is required for DNA synthesis of few kilobases and that the latter was necessary for template switching. Surprisingly, we observed in pol32D survivors an extremely high level of chromosome III circularization, even in Ura+ survivors that likely repaired the DSB on chromosome VII by GC or NHEJ (Figure 7 and Figure S10). This would mean that DSBs induced at HMR could not be repaired by HR with MATa-inc sequences in pol32D cells. Indeed, preferential formation of crossovers between MAT and HMR would lead to the extrusion of genes essential for viability, whereas crossovers between HMR and HML would lead to the formation of stable circular chromosomes. Preferential processing of recombina- tion intermediates into crossovers in pol32D mutants have been reported in other studies, in which BIR events were aborted and resulted in half-crossovers [22,23]. Mus81 and Yen1 redundantly promote BIR We determined the role of SSEs in the generation of chromosomal rearrangements using our assay as the goal to identify the nucleases that are required during BIR. Overall, we observed a significant decrease of chromosomal rearrangements in mus81D single mutants that was aggravated in mus81D yen1D mutants (Figure 4A). We have confirmed that the frequency of translocants only decreased in the mus81D yen1D mutants in the HMR-inc background (Figure 4B), in which BIR, and not BIR/ SSA, is expected to occur. This indicates that Mus81 may play a role in BIR/SSA and that Mus81 functions can be fully taken over by other proteins during BIR. However, our data show that both Mus81 and Yen1 carry out redundant or equivalent activities, which are needed for BIR completion. Mus81 and Yen1 have already been implicated in DSB repair by recombination but not directly in BIR. Mus81 has been shown to act at replication forks. It has been proposed that Mus81 could cleave stalled forks but also to participate in recombination-mediated repair of cleaved or collapsed forks to allow their restart in yeast and humans [32– 34,57]. Mus81 is also required in humans for telomere recombi- nation to allow proliferation of telomerase-negative cancer cells [58]. Formally, both mechanisms of replication fork restart and telomere recombination are equivalent to BIR. Yen1 roles in PLOS Genetics | www.plosgenetics.org 13 September 2012 | Volume 8 | Issue 9 | e1002979