ADH4-59#1 GCGCGCGGTACCGAATTCAAACCGCTGAT- TACATCAAA and ADH4-59#2 GCGCGCAGATCTATC- GATCTCGAGTCTAGACTAGACCAGTAGCAGCAGTC, and ADH4-39#1 GCGCGCAGATCTGCTAGCACTAGTGGATC- CCTTAGTCGCTGCATACAAAG and ADH4-39#2 GCGCG- CGAGCTCGAATTCGCACACGCATAATTGACGTT. These two fragments were cloned by the gap repair method in pBluescript II(SK+) previously digested by KpnI and SacI to create pBP99 plasmid. A BglII-BamHI URA3 containing fragment from sp392 plasmid [71] was then cloned in pBP99 digested with BglII to create pBP102. Finally, the HMR fragment was amplified from genomic DNA with the primer pair HO-HMR-Hind3 GCGCGCAAGCTT- CAACCACTCTACAAAACCAAAACCA and HO-HMR-Nhe1 GCGCGCGCTAGCAGAAGAAGTTGCAAAGAAATGTGGC and cloned into pBP102 after digestion with HindIII and NheI, to create pBP102-HO. pBP102-HO was linearized with PvuII and transformed into yeast. Integration was selected by uracile prototro- phy and verified by PCR and Southern analysis. Determination of survival and translocation frequencies Yeast cells were grown in yeast extract-peptone-adenine- dextrose (YPAD) until reaching the exponential phase of growth, appropriately diluted with H2O and plated on synthetic complete (SC) medium containing either 2% glucose or 2% galactose as a carbon source. Survivor colonies on galactose-containing plates were then restreaked on YPAD plates and replica-plated on SC plates containing 5-FOA (USBiological), a drug that generates a toxic metabolite in Ura+ cells, or on SC plates lacking uracil. Frequencies were calculated as follows: survival frequency = cfu galactose/cfu glucose; translocants frequency = (cfu galactose Ura25-FOAr+(cfu galactose Ura+5-FOAr)/2)/cfu glucose. 96 to 288 survivor colonies, recovered from 2 to 3 independent induction experiments, were analyzed for each strain tested. Statistical analysis was performed using the x2 test with Yates’ correction. Kinetic analysis of BIR intermediates by PCR and Southern analysis Yeast cells were grown at 30uC in liquid YPAD until reaching the exponential phase of growth, washed twice with synthetic complete medium SGL (3% glycerol, 2% lactate) and cultured overnight in SGL until reaching an OD600 nm<0.5 when galactose was added at a final concentration of 2%. Cells were taken at different times after galactose induction and genomic DNA was extracted in agarose plugs according to standard procedures. Agarose plugs were incubated twice in 200 ml 16 b-Agarase I reaction buffer for 30 min, melted at 65uC for 10 min, equilibrated at 42uC for 15 min and treated with b-Agarase I (New England BioLabs) at 42uC for 1 h before PCR amplification. These were performed with 250 ng of genomic DNA (estimated with Nano- Drop, Thermo Scientific) in a total volume of 30 ml in the following conditions: 16 Phusion HF buffer, 200 mM each dNTP, 0.6 U Phusion DNA polymerase (Finnzymes), 0.5 mM each primer. Samples were denatured for 45 s at 98uC, then cycled 25–35 times with 20 s denaturation (98uC), 30 s annealing (57uC) and 45 s extension (72uC) followed by a final extension step of 5 min at 72uC. PCR was performed with primer p7 GCACACGCATAATT- GACGTT and primers p3-M GAAGACTTGTGGCGAAGA, p3- R CCAACATTTAGGAAAAAACG or p3-L CGGATGGCA- CAAGGAACACGCATTT. Control PCR was performed with primers corresponding to ACT1 locus, ACT1up TTCACGCT- TACTGCTTTTTTC and ACT1low CAAGGCGACGTAACA- TAGTTT. PCR products were subjected to gel electrophoresis in 0.8% agarose and stained with ethidium bromide. Instead of b- Nuclease Processing of BIR Intermediates Agarase I treatment, plugs were digested with 30 U of EcoRV restriction enzyme for 5 h at 37uC and loaded in a 1% agarose gel for Southern analysis. Electrophoresis was run at 80 V for 16 h30 and DNA was transferred into Hybond-XL membranes (GE Healthcare) in alkaline conditions. Membranes were probed with dCT32P-labelled PCR fragments obtained with ADH4-39#1 and ADH4-39#1 primers (7L probe). Quantification of DNA signals was made relative to the total DNA of each lane and was performed using ImageGauge 4.2 (Fujifilm) program. Pulsed-field gel electrophoresis (PFGE) analysis For each strain, 28 to 84 independent Ura- 5-FOAr survivor colonies were grown in 2,5 ml of YPAD medium overnight at 30uC. Agarose plugs containing chromosomal DNA were made according to the manufacturer’s instructions (Bio-Rad). AsiSI digestion was performed incubating agarose plugs twice in 1 ml 16 NEBuffer 4 for 30 min and digested in 200 ml 16 NEBuffer 4 with 30 U of AsiSI restriction enzyme for 5 h (New England Biolabs). Agarose gels (0.9%) were run in a Bio-Rad CHEF MapperXA apparatus for 16 h at 6 V/cm with a switch time of 70 s and for an additional 12 h at 6 V/cm with a switch time of 120 s. Then, gels were stained with ethidium bromide and DNA was transferred into Hybond-XL membranes (GE Healthcare) in alkaline conditions. Membranes were probed with dCT32P-labelled PCR fragments obtained with primers ADH7#1 TGTTGGCTAAAGCTATGG and ADH7#2 TTCTTCGCTGATCGG (3R1 probe), ARS315#1 AAACCAGTCTTTAACCGCCATAATG and ARS315#2 CA- GAGCCCAAGAGATAGCCGAACTT (3R2 probe), and with primers HML+HMR-F CAAACATCTTAGTAGTGTCTGAG- GA and HML+HMR-R CTGTAATTTACCTAAGTTACCA- GAG (X probe). Chromosomal rearrangements different from T7/3-MAT or T7/3-HMR translocations or circular chromosomes III and revealed by the PFGE analysis were not included in the statistical analyses. Supporting Information Figure S1 Survival frequency to DSB induction in the WT and SSE mutants. Graphical plotting of survival frequency of each strain tested. See Material and Methods for more details. {, frequency,1024. Error bars represent standard deviations. (TIF) Figure S2 PFGE analysis of translocations in mixed Ura+ 5- FOA-resistant colonies. Translocations between chromosomes VII and III were detected by PFGE followed by Southern analysis using the 7L (A) and the 3R1 (B) probe. Chromosomal DNA samples were extracted from previously separated Ura+ and 5- FOA-resistant cells. Black arrows indicate the positions of the T7/ 3-HMR translocations and of the linear chromosome III (Chr. III). (TIF) Figure S3 DNA invasion at HMR causes its cleavage. (A) Schematic representation of chromosomes VII and III in our assay. Localization of the 7L and X probes is indicated. (B) Detection of HO cleavage on chromosome VII, as monitored by Southern analysis using the 7L probe in the WT and hmlD matDYZ strains. DNA samples were extracted at different intervals after HO induction with galactose. Positions of the bands corresponding to the uncut and cut chromosome VII (chr. VII) are indicated. (C) (D) Detection of HMR cleavage product, as monitored by Southern analysis using the X probe in the WT and hmlD matDYZ strains and the hmlD matDYZ strain without HO cut site in chromosome VII. DNA samples were extracted at different intervals after HO induction with galactose. Quantification of PLOS Genetics | www.plosgenetics.org 16 September 2012 | Volume 8 | Issue 9 | e1002979