Nuclease Processing of BIR Intermediates Figure 6. Template switching is affected in mus81D slx4D yen1D but not in rad1D mutants. (A) Appearance of BIR repair products, as monitored by Southern analysis, in WT, rad1D and mus81D slx4D yen1D strains. Experiments were performed as described in Figure 2C. (B) Quantification of BIR product accumulation. Quantification results for chromosome VII cleavage (DSB), 7/3-HMR and 7/3-HML BIR intermediate accumulation are shown in percent. (C) Quantification of 7/3-MAT/7/3-HMR BIR intermediate ratios 24 h after HO induction. Mean values and standard deviations for 2–3 independent experiments are shown. (D) Appearance of BIR repair products, as monitored by Southern analysis, in HMR- inc rad1D, HMR-inc mus81D slx4D yen1D and HMR-inc slx4D strains. (E) Quantification of BIR product accumulation. Quantification results for chromosome VII cleavage (DSB), 7/3-HMR, 7/3-HML and 7/3-MAT BIR intermediate accumulation are shown in percent. Mean values and standard deviations for 2–3 independent experiments are shown. A representative Southern analysis is shown for each genotype analyzed. 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. Bands marked with a red asterisk likely result from partial digestion. GAL, galactose; h, hours. doi:10.1371/journal.pgen.1002979.g006 These results confirmed that the cleavage of the HMR locus upon its invasion in the HMR WT strain facilitated the formation of T7/ 3-HMR translocations as opposed to T7/3-MAT translocations. Additionally, possible cleavage of HMR upon the passage of the BIR fork initiated at MATa-inc impaired the formation of T7/3- MAT translocations. Nevertheless, slx4D single mutants did not show any increase of T7/3-MAT translocations and mus81D slx4D yen1D mutants showed a higher increase of T7/3-MAT translo- cations than rad1D mutants (Figure 5B). We hypothesized that this was due to a processing defect of BIR intermediates formed at MATa-inc that impeded subsequent template switching to HMR and HML. To explore this possibility, we analyzed the kinetics of appearance of BIR intermediates in mus81D slx4D yen1D mutants, as well as in the rad1D and slx4D single mutants, in the HMR-inc background (Figure 6D, 6E). In HMR-inc strains, BIR/SSA does not occur and the accumulation of 7/3-HML intermediates serves as an indicator of the efficiency of template switching during BIR. In contrast to the HMR-inc rad1D and HMR-inc slx4D mutants, which did not show any significant difference compared to the HMR-inc WT strain, 7/3-HML intermediates were reproducibly not detected at all time points in HMR-inc mus81D slx4D yen1D mutants. Accumulation of 7/3-HMR and 7/3-MAT intermediates was also significantly lower in the latter strain, probably reflecting the BIR defect previously observed in HMR-inc mus81D yen1D cells. Altogether, these data indicate that DSB repair was altered in cells lacking Rad1, defective in BIR/SSA, and in cells lacking all three SSE factors Mus81, Slx4 and Yen1 that show a defect of template switching during BIR. Chromosome III circularization and crossover activity Among the chromosomal rearrangements generated in our strains, circularization of chromosome III occurred at a high frequency. No chromosome III circularization was observed in the control HMR-inc WT strain (Figure S7B), demonstrating that HMR cleavage induced this secondary recombination event (type c, Figure 3). HMR cleavage and translocations via BIR/SSA would leave chromosome III with a one-ended DSB. Thus, circularization of chromosome III is thought to occur by a recombination event that ended in a half-crossover, characterized by a reciprocal exchange between HMR and HML that caused the loss of the chromosome III left telomere and the formation of a chromosome circle (Figure 7A). Circularization of chromosome III is not mandatory for survival of BIR/SSA-mediated translocants since the translocation can co-segregate with the uninvolved chromatid of chromosome III (type b, Figure 3). Therefore, we took advantage of chromosome III circularization events to investigate the ability of SSE mutant cells to produce half- crossovers. PFGE analyses allowed us to detect circular chromosome III in all strains as signals appearing in the wells (Figure 7B and Figures S8, S9). In addition to the well signal, we also detected in some strains a faint signal for the truncated linear chromosome III. This faint signal corresponds in size to the circular chromosome III cleaved by AsiSI (Figure 1E). Since we did not find any linkage between the presence of this signal and a particular genetic background, we conclude that this is likely due to breakage occurring during DNA extraction or PFGE. To get further insight into the mechanism that gave rise to circular chromosomes, we have evaluated the percent of translocants that contained circular versus linear chromosomes III for each genotype (Figure 7C). Whereas about 50% of WT translocants contained a circular chromosome III, this percent increased significantly to 88% (n = 59, x2, p,0.01) in pol32D mutants (Figure 7B, 7C). This result is concordant with previous observations showing that pol32D defects led to D-loop processing during BIR that generated half- crossovers [22,23]. Among the SSE mutants tested, the proportion of translocants containing circular chromosomes III decreased significantly to 33% and 30% in mus81D and slx1D mutants, respectively (x2, p,0.05) (Figure 7B, 7C). In contrast to this observation, the percentage of translocants containing circular chromosomes III increased significantly up to 72% in mus81D slx4D double mutants (x2, p,0.01) (Figure 7B, 7C). These results suggest that Mus81 and Slx4 have different roles regarding crossover formation. Additionally, we observed that yen1D mutation suppressed the increase detected in mus81 slx4D mutants, suggesting that Yen1 action may be possible only in the absence of Slx4. We conclude that Slx4, which has been described as acting as a platform with different nuclease complexes [52], might regulate Mus81 and Yen1 accessibility to recombination interme- diates or their nuclease activity to generate crossovers. We also analyzed chromosome III circularization in Ura+ survivors, which did not contain translocations and likely performed DSB repair by GC or NHEJ (Figure S10). Analogously to what we observed in the Ura2 translocants, 36% of WT Ura+ survivors contained a circular chromosome III and this percentage increased significantly to 90% in pol32D mutants (Figure S10E). This suggests that chromosome III circularization does not depend on the translocation event and happens similarly in all survivors whether Ura+ or Ura2. Finally, we calculated the efficiency of chromosome III circularization for each genotype (Figure 7D). Pol32 appears to be required for efficient chromosome III circularization since the calculated efficiency went down signifi- cantly from 22% in WT to 10% in pol32D translocants (x2, p,0.05) (Figure 7D). This probably explains the low survival of pol32D mutants to the DSB induction (Figure S1). Similarly, we observed a significant decrease in the efficiency of chromosome III circularization in mus81D yen1D mutants (Figure 7D). This is consistent with the conclusion that Mus81 and Yen1 play an important role in the formation of the majority of circular chromosomes III. Discussion We developed an assay to study the molecular mechanisms that lead to complex chromosomal rearrangements upon induction of a PLOS Genetics | www.plosgenetics.org 11 September 2012 | Volume 8 | Issue 9 | e1002979