Nuclease Processing of BIR Intermediates Figure 8. BIR model showing SSEs involvement in template switching and half-crossovers. After invasion of the homologous template by one end of the DSB, a D-loop is formed and extended by the priming of DNA synthesis from the invading strand 39 end. The D-loop can be specifically cleaved by Mus81 or branch migrated to create a HJ cleavable by Yen1, to establish a replication fork. Extensive replication, which requires Pol32, would complete BIR and generate a non-reciprocal translocation (NRT). Alternatively, [a] cleavage of the replication fork by Mus81-Slx4-Yen1 would allow re-invasion of the same template or template switching. [b] cleavage of the replication fork by Mus81-Slx4-Yen1 would terminate the BIR event, causing a half-crossover (NRT). RF, replication fork, HJ, Holliday junction. doi:10.1371/journal.pgen.1002979.g008 template switching and half-crossovers, which generate complex chromosomal rearrangements and prolonged cycles of genomic instability. Such events are thought to be at the origin of various genomic disorders and cancer development [24,69]. Materials and Methods Yeast strains and plasmids All Saccharomyces cerevisiae yeast strains used in this study are in W303-1a background (his3-11, 15 leu2-3, 112, trp1-1 ura3-1 ade2-1 can1-100 rad5-535) [70] and harbor MATa-inc, ade3D::gal-HO and leu2D::SFA1 alleles [53]. The MATa-inc HMR-inc strain was obtained by mating-type switching inducing HO expression in a MATa HMR-inc strain. Independent survivors to HO expression were selected and the MAT and HMR loci were sequenced to verify the MATa-inc HMR-inc genotype. Deletion mutants were either obtained by the PCR-based gene replacement method (verified by PCR and Southern) or by genetic crosses (verified by tetrad analysis). Deletion of MAT is only partial (matDYZ) because of the presence of other genes overlapping with MAT. Only the Y and Z sequences, containing the HO cut site, have been removed. Insertion of a HO-cleavable 240-bp HMR fragment at the ADH4 locus has been conducted as follows. Two 59 and 39ADH4 fragments were amplified by PCR with the following primer pairs PLOS Genetics | www.plosgenetics.org 15 September 2012 | Volume 8 | Issue 9 | e1002979