Nuclease Processing of BIR Intermediates Figure 1. Description of the assay used in this study. (A) Schematic representation of chromosomes VII (black) and III (blue) in the haploid strain; X region (x, black box), Ya region (a, red box), Ya region (a, blue box) and Z1 region (z, green box) are indicated on chromosome III, as well as the Ya-Z1 fragment on chromosome VII. See text for more details. (B) Determination of translocation events. Cells were plated on solid synthetic medium containing galactose and survivor colonies were restreaked on rich YPAD medium containing glucose and subsequently replica-plated on SC+5-FOA and SC-ura. (C) (D) Translocations between chromosomes VII and III were detected by PFGE followed by Southern analysis using the 7L and the 3R1 probe (see panel A). Chromosomal DNA samples were extracted from previously selected Ura2 5-FOAr survivor colonies. (E) Detection of circular chromosome III after PFGE and Southern analysis. Chromosomes III that were not hybridized with the 3R1 probe were detected in the gel wells using 3R2 probe (see panel A). The DNA samples corresponding to circular chromosome III were digested with AsiSI to linearize chromosome III. C, control strain without translocation. (F) PFGE followed by Southern analysis using the 3R2 probe of Ura2 5-FOAr survivors in hmlD strain. Black arrows indicate the positions of the T7/3-MAT and the T7/3-HMR translocations and the circular and linear chromosomes III (Chr. III). PFGE, pulse-field gel electrophoresis. 5-FOAr and 5-FOAs refers to colonies either resistant or sensitive to 5-FOA. (G) Schematic representation of the predominant chromosomal rearrangements observed in this study. doi:10.1371/journal.pgen.1002979.g001 Interestingly, some survivors lacked linear chromosome III at its expected size in the PFGE analysis (Figure 1D, 1E). When we used a probe specific to the region between MAT and HMR loci (3R2 probe, Figure 1A), chromosome III was detected in the well, consistent with a particular structure that did not allow it to enter the gel (Figure 1E). One possibility was that chromosome III became circular, as had been observed more than three decades ago [47,48]. To test this, we digested chromosomal DNA with AsiSI, which cuts chromosome III at a unique inner location. Indeed, AsiSI digestion released chromosome III into the gel (Figure 1E). Circular chromosomes III have been shown to be the product of recombination between unrepressed HMR and HML loci, which share extensive homology in the X and Z1 regions (Figure 1A) [47]. We observed that the size of the AsiSI linearized chromosome was concordant with a chromosome III that would have lost all subtelomeric sequences located beyond the HM loci. To further demonstrate that circular chromosomes III occurred by recombination between HMR and HML, we assayed whether chromosome III circularization would be seen in hmlD cells. We observed that, in the absence of HML locus, all survivors contained a linear chromosome III at its expected size (Figure 1F). Chromosome III circularization pushed us to investigate the occurrence of a DSB at HMR. HMR is naturally packed into heterochromatin to repress its expression and also to impede cleavage by HO [44]. We asked if HMR invasion would release its repression and cause HO cleavage. We first assayed HMR cleavage by Southern at several times after induction of chromosome VII cleavage by HO and did not observe any cuts in the WT (Figure S3C). We only detected HMR cleavage in a hmlD matD strain, in which HMR cleavage represented about a third of chromosome VII cleavage by HO (17% versus 46% after 3 h of induction; Figure S3B, S3C). Indeed HMR cleavage was dependent on cleavage at chromosome VII since no cuts were observed in the hmlD matD strain without HO cut site on chromosome VII (Figure S3D). We concluded that the induction of a single DSB by HO in chromosome VII permitted another less efficient DSB in chromosome III at the HMR locus as a secondary event. The efficiency of HMR cleavage is expected to be even less than 17% in the WT strain since the MATa-inc locus is also available as a template for strand invasion in this strain. HMR cleavage likely induced chromosome III circularization, which we observed in 50% of translocants in the WT strain. Apart from T7/ 3-MAT, T7/3-HMR translocations and chromosome III circular- ization, we also observed rare types of rearrangements of chromosome VII and III whose nature has not been addressed in this study. Together, these results demonstrate that complex chromosomal rearrangements are occurring at a high frequency in our experimental system, allowing us to investigate the molecular and genetic bases of these events. DSB repair involves template switching between MATa- inc, HMR, and HML We performed kinetic experiments to follow the recombination intermediates that gave rise to chromosomal rearrangements using our system. First, a PCR-based assay was used to monitor new DNA synthesis primed from the 39 end of the invading strand of chromosome VII. Genomic DNA was extracted at different times after HO induction and PCR was made with one primer specific to chromosome VII (p7, Figure 2A), proximal to the DSB, and primers specific to each potential template used for recombination, MATa-inc and HMR (p3-M and p3-R, Figure 2A). In WT cells, 35 cycles of PCR amplification permitted to detect products that most likely correspond to newly synthesized DNA fragments at both MAT and HMR loci 1 h after DSB induction (Figure 2B). Using more quantitative conditions, we detected the same products after 25 cycles of PCR amplification at 2 h of DSB induction (Figure S4). The amount of products increased over time. We then monitored the appearance of repair intermediates directly by Southern analysis of genomic DNA digested by EcoRV, using a probe specific to chromosome VII, proximal to the DSB site (7L probe). We detected recombination intermediates between chro- mosome VII and chromosome III at HMR (7/3-HMR, Figure 2C, left), which most likely correspond to newly synthesized DNA fragments 5 h after DSB induction. At MAT, we observed recombination intermediates 24 h after DSB induction (7/3- MAT, Figure 2C, left). Southern analysis also detected an unexpected band that corresponded in size to chromosome VII fused to chromosome III sequences at the HML locus (7/3-HML, Figure 2C, left). We confirmed this assumption by re-probing the Southern membrane with a probe specific to HML (data not shown). The 7/3-HML band most likely corresponds to newly synthesized DNA fragments primed from chromosome VII DSB end at HML and appeared concomitantly with 7/3-HMR intermediates 5 h after DSB induction (Figure 2C). We monitored what was most likely new DNA synthesis primed from the 39 DSB end of chromosome VII invading HML by PCR. We detected intermediates 2 h after DSB induction (p7/p3-L, Figure 2B, Figure S4). The DSB end of chromosome VII assayed by PCR and Southern only shares sequence homology with MATa-inc and HMR. Hence, the signals detected at HML would be the consequence of a template switching from MATa-inc or HMR to HML after duplication of the Z1 region that is common to the three loci. In a similar way, template switching could occur between MATa-inc and HMR. 7/ 3-HMR and 7/3-HML intermediates detected by Southern appeared about 3–4 h after detection of priming of the 39 invading DSB end by PCR (Figure 2B, 2C). This difference could be due to the difference of sensitivity between the two techniques. Alternatively, this could reflect a transition between the elongation of the 39 invading DSB end, a step that is common to GC and BIR, and the establishment of an active replication fork required PLOS Genetics | www.plosgenetics.org 4 September 2012 | Volume 8 | Issue 9 | e1002979