Nuclease Processing of BIR Intermediates Figure 3. Various pathways give rise to chromosomal rearrangements. Schematic representation of the different types of repair giving rise to the chromosomal rearrangements scored in this study. Repair of DSBs in G2 can occur either by BIR, BIR/SSA following HMR cleavage with/without circularization of chromosome III, gene conversion (GC) or erroneous NHEJ. The repair types of only one DSB are depicted for simplification. The other DSBs are repaired via either one of the different types. VII, VII9 and III, III9 indicate chromosome VII and chromosome III sister-chromatids, respectively. U, URA3; M; MAT; R, HMR; L, HML; M/R, MAT or HMR. See text for more details. doi:10.1371/journal.pgen.1002979.g003 No BIR intermediate could be detected in HMR-inc pol32D cells (Figure 4C, 4D). In HMR-inc mus81D yen1D cells, a decrease of BIR intermediates was reproducibly observed at all time points (Figure 4C, 4D). We concluded that Mus81 and Yen1 are both required for promoting efficient Pol32-dependent BIR. Template switching is affected in structure-selective endonuclease mutants We confirmed by PFGE that all 5-FOA-resistant survivors analyzed in mutant backgrounds contained chromosome translo- cations between chromosome VII and chromosome III (Figure 5A and Figures S5, S6). Notably, a very low amount of T7/3-MAT translocations (8%) were recovered in WT cells (Figure 5B). In pol32D cells, no T7/3-MAT translocation was recovered (n = 59, x2, p,0.05), showing that this type of translocation has a complete dependency on Pol32 (Figure 5A, 5B). Among the nuclease mutants, rad1D showed a significant increase of T7/3-MAT translocations, up to 20% of total translocations (2.5-fold, n = 59, 2 x , p,0.01), which was not observed in double mutants with mus81D and slx1D (Figure 5A, 5B). The concomitant absence of Mus81, Slx4 and Yen1 engendered an even higher increase (3.1- fold) of T7/3-MAT translocations that reached up to 25% of total 2 translocations (n = 58, x , p,0.01) (Figure 5A, 5B). In kinetic experiments, both 7/3-HMR and 7/3-HML intermediates accu- mulated in the rad1D and mus81D slx4D yen1D mutants with kinetics clearly delayed (2–3 h) respect to the WT, demonstrating a defect of repair in these mutants (Figure 6A, 6B). At 24 h, signals for 7/3-HMR, 7/3-HML and 7/3-MAT that likely correspond to final repair products were detected in all strains. Notably, we observed a clear increase in the 7/3-MAT/7/3-MAT ratio, up to 24% and 29% in rad1D and mus81D slx4D yen1D, respectively, compared to the 15% seen in the WT (Figure 6C). This PLOS Genetics | www.plosgenetics.org 7 September 2012 | Volume 8 | Issue 9 | e1002979