Alactose to liquid cultures. Following galactose addition, yeast cultures have been incubated for four h to be able to rapidly induce the DSBs in G1accumulated cells. Just after this incubation time, appropriate dilutions had been plated onto full galactose-containing media with (SGal) or without (SGal-Leu) Conglobatin Inhibitor leucine. Cell survival was determined by dividing the number of colonies developing on SGal after DSB repair by the number of colonies growing on SC just before DSB induction. The frequency of translocations was determined by dividing the amount of colonies expanding on SGal-Leu by the amount of colonies growing on SC (total cells). This parameter was made use of as a reference value to examine distinct strains. To determine recombination frequencies in the repair of DSBs generated in cis we employed a previously reported yeast genetic assay [34]. Briefly, suitable dilutions of cells from overnight cultures in glycerol-lactate with no uracil have been Chlorfenapyr Protocol spread on glucose- and galactose-containing plates. Survivor colonies on galactose-containing plates were replica-plated on SC plates containing 5-FOA (USBiological), to discriminate in between Ura2 and Ura+ cells. The frequency of DSB repair involving the loss in the URA3 gene was determined by dividing the amount of colonies increasing on SC+5FOA by the amount of colonies expanding on SC. Statistical significance of translocation frequencies in mutant strains was evaluated using the Mann-Whitney test compared to wildtype cells (in mutant strains yku70D, pol4D, tel1D and tel1D pol4D), or compared to pol4D [POL4] cells (in pol4D cells overexpressing mutant Pol4 versions). The distribution of repair events obtained within the unique mutant strains was in comparison with that of wild-type strain utilizing the Chi-square test. The distribution of repair events obtained in pol4D cells overexpressing mutant Pol4 versions was compared to that of pol4 [POL4] strain using the same test.Amino acid sequence comparisons and 3D-modellingMultiple alignment of the 3 Saccharomyces Pol4 DNA polymerases was done using MULTALIN (http://multalin. toulouse.inra.fr/multalin). Pol4 amino acid sequence was modeled making use of human Poll PDB coordinates and Swiss-Model application (http://swissmodel.expasy.org). For tridimensional structure extrapolations, we compared this Pol4 model with crystal structure of human Poll within a ternary complex with a 1-nt gapped DNA substrate and the incoming nucleotide (PDB code:1XSN) [48]. This was obtained in the Protein Data Bank (http://rcsb.org/ pdb). Pol4-Thr540 residue and the corresponding point mutation was identified by utilizing PyMol computer software (http://pymol.org/).MiscellaneousChromosomal breakpoint analysis by PCR and DNA sequencing, and molecular karyotyping of Leu+ translocants by pulsedfield gel electrophoresis have been performed as previously described [35,52]. Breakpoint sequences from all sequenced Leu+ translocants are shown in Figure S2.Supporting InformationFigure S1 Molecular karyotype of wild-type Leu+ translocants. (Upper) Scheme in the assay. (Lower) PFGE analysis of 12 independent wild-type translocants. Parental strain (P) is shown as a reference. Gels were stained with ethidium bromide (left) and analyzed by Southern making use of an HYG specific probe (right). Electrophoretic mobility of organic yeast chromosomes is indicated around the left. Right after DSBs induction and Leu+ choice, two newPLOS Genetics | plosgenetics.orgPol4-Mediated Chromosomal Translocationstranslocated chromosomes is usually detected (tIII/XV and tXV/III, marked with.