Author Summary Genome rearrangements consisting of non-reciprocal translocations (NRTs) seem to play an important role in carcinogenesis in humans. They are likely caused by intracellular mechanisms that are normally committed to repair breaks occurring in the DNA molecule. Failure of faithful repair of DNA double-strand breaks (DSBs) often leads to chromosomal rearrangements when repair occurs within repeated genomic regions. The break-induced replication (BIR) pathway of DSB repair is a major source of complex chromosomal rearrangements, the latter occurring when BIR involves template switching between dispersed repeated sequences. Given the deleterious consequences of such events for genomic stability, it is of great significance to understand the molecular bases of BIR. Here, we examined the role of different DNA nucleases in chromosomal rearrangements and uncovered the functional involvement of the structure-selective endonu- cleases (SSEs) subunits Mus81, Yen1, and Slx4 at different steps during BIR. Our work provides new clues to understand the origin of NRTs and the role of SSEs in their generation. cells have evolved a set of DNA structure-selective endonucleases (SSEs) that possess different substrate specificity for various DNA branched molecules during HR, such as D-loops, replication forks, flaps and Holliday junctions (HJs). Conversion of a D-loop into a replication fork during BIR may require the endonucleolytic resolution of a single Holliday junction [16,25]. In budding yeast, Mus81-Mms4 and Yen1 are the only nuclear enzymes capable of cleaving intact HJs with high efficiency in vitro [26,27]. In vivo, their functions seem to overlap during DSB repair [28–31]. Notably, Mus81-Mms4 is required for recombination-mediated DNA repair at replication forks [32–34] and has been shown to play a role in BIR intermediate processing [23]. Additionally, the Slx1– Slx4 nuclease complex may cleave perturbed replications forks [35–37]. Loss of Slx1–Slx4, as loss of Mus81-Mms4, increases the number of gross chromosomal rearrangements in yeast [38]. Slx4 also acts independently of the Slx1 catalytic subunit to interact with several other factors. For instance, Slx4 binds to the 39 flap endonuclease complex Rad1–Rad10 to facilitate the removal of non-homologous tails during HR [39,40]. Rad1–Rad10 complex, which functions in nucleotide excision repair (NER) as well as in DSB repair, has also been involved in the formation of translocations in yeast through a single-strand annealing (SSA) mechanism [41,42]. Together, these SSEs form a complex network to ensure genome stability but their specific roles and their interactions at recombination structures remain unclear. In this study, we used a new assay that generates chromosomal rearrangements after DSB repair using dispersed repeated sequences. Chromosomal rearrangements involved multiple rounds of template switching and some events ended in half- crossovers, generating NRTs. We investigated the role of SSEs in the processing of BIR intermediates combining mutations of Mus81, Rad1, Yen1, Slx1 and Slx4. Our results show that these SSEs act at multiple steps during BIR. First, we uncovered that Mus81 and Yen1 function to allow efficient BIR, thus causing translocations. In the absence of Mus81, Yen1 and Slx4, we observed that template switching during BIR decreased signifi- cantly. Altogether, our results led to new insights into the BIR mechanism and the functional role of SSEs in chromosomal rearrangements. Nuclease Processing of BIR Intermediates Results A new assay for DSB–induced chromosomal rearrangements We designed an experimental system using DNA repeated sequences dispersed over two yeast chromosomes for the analysis of chromosomal rearrangements induced by a single DSB. We took advantage of the presence of the MAT, HMR and HML loci on chromosome III, involved in mating-type switching. The MAT locus is composed of five regions called W, X, Y, Z1 and Z2 [43]. MATa differs from MATa by the Ya and the Ya sequences, respectively. MATa shares Ya with HMR and MATa shares Ya with HML. Together, MAT, HMR and HML share two homologous regions flanking the Y sequences, termed X and Z1 (see Figure 1A). All three loci contain a cleavage site for the HO endonuclease at the junction between the Y and Z1 regions, but only the MAT locus is susceptible to being cleaved upon HO expression because of the active repression of HMR and HML loci [44]. The strains used in this study harbor a MATa-inc mutation, a G to A substitution at position Z1–2, which impedes HO cleavage at MAT [45,46]. The HO endonuclease gene under control of the GAL1 inducible promoter was integrated at the ADE3 locus and a 240-bp Ya-Z1 fragment containing a HO-cleavable site, along with the URA3 marker, was inserted in the chromosome VII left arm at the ADH4 locus (Figure 1A). Upon galactose addition to the culture medium, HO would cleave this Ya-Z1 fragment asym- metrically into a centromeric 198-bp fragment and a telomeric 42- bp fragment (Figure 1A). To assay DSB repair in this system, we plated cells on a synthetic medium (SC) with either 2% glucose or 2% galactose to assay survival after HO expression and successive breakage of chromosome VII. The WT strain exhibited a survival frequency of 82% (Figure S1), demonstrating efficient DSB repair. We restreaked survivor colonies from galactose-containing plates on glucose medium to repress HO expression. Then, survivors were concomitantly replica-plated on media lacking uracil and on media containing 5-FOA, a drug that generates a toxic metabolite in Ura+ cells, to distinguish between the colonies that maintained or lost the URA3 marker of chromosome VII (Figure 1B). We recollected Ura2 5-FOA-resistant (20%), Ura+ 5-FOA-sensitive (12%), and Ura+ 5-FOA-resistant colonies (68%). The latter contained both Ura+ and Ura2 cells and may result from differential repair of two DSBs generated on sister chromatids during the S or G2 phase of the cell cycle. To analyze mixed Ura+ 5-FOA-resistant colonies, we first separated Ura+ cells from 5- FOA-resistant cells by restreaking colonies on media lacking uracil or containing 5-FOA. Further analyses of 5-FOA-resistant and Ura+ cells were performed by pulse-field gel electrophoresis (PFGE) to look for chromosomal translocations. Southern analysis using a probe specific to chromosome VII, proximal to the DSB site, and a probe specific to the region between HMR locus and chromosome III telomere (7L probe and 3R1 probe, respectively, Figure 1A) revealed the presence of non-reciprocal translocations (NRTs) between chromosome VII and chromosome III in all 5- FOA-resistant cells (Figure 1C, 1D, and Figure S2A, S2B). NRTs were rarely observed in Ura+ cells (Figure S2B). Given a survival rate of 82%, the frequency of translocants (frequency of 5-FOA- resistant survivors among the whole population that did not undergo DSB induction) was 44% in the WT strain. We mainly observed two types of NRTs, which contained chromosome III sequences starting from the MAT or HMR loci to the telomere fused to chromosome VII at the break site (termed T7/3-MAT and T7/3-HMR translocations for more clarity, Figure 1G). PLOS Genetics | www.plosgenetics.org 2 September 2012 | Volume 8 | Issue 9 | e1002979