Es and Haber,).This was accomplished by replacing to prevent repair by single strand annealing (Pa sequences upstream and downstream of ARG with natMX (Goldstein and McCusker,) and K.lactis TRP sequences (Stark and Milner,) respectively (see Supplementary file legend for specifics).The resulting argVRS and argVRS inserts share .kb of homology.VDE ordinarily exists as an intein within the constitutivelyexpressed VMA gene (Gimble and Thorner,), resulting in low levels of DSB formation in presporulation cultures (information not shown), almost certainly as a consequence of tiny amounts VDE incidentally imported towards the nucleus in the course of mitotic development (Nagai et al).To further restrict VDE DSB formation, strains have been constructed in which VDE expression was copperinducible.These strains include the VMA allele (Nogami et al), which delivers wild form VMA function, but lacks the VDE intein and is resistant to cleavage by VDE.To produce strains in which VDE expression was copperinducible, VDE coding sequences on an EcoRI fragment from pY (Nogami et al); a generous gift from Dr.Satoru Nogami and Dr.Yoshikazu Ohya) had been inserted downstream with the CUP promoter in plasmid pHG, which includes the kanMX selectable marker in addition to a PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/21493362 kb CUP promoter fragment (Jin et al), to make pMJ, which was then integrated at the CUP locus.Medhi et al.eLife ;e..eLife.ofResearch articleGenes and ChromosomesSporulationYeast strains had been grown in buffered liquid presporulation medium and shifted to sporulation medium as described (Goyon and Lichten,), except that sporulation medium contained uM CuSO to induce VDE expression.All experiments have been performed at .DNA extraction and analysisGenomic DNA was ready as described (Allers and Lichten,).Recombination merchandise have been detected on Southern blots containing genomic DNA digested with HindIII and VDE (PISceI, New England Biolabs), using particular buffer for PISceI.Samples were heated to for min to disrupt VDEDNA complexes just before loading; gels contained .agarose in mM Tris Borate mM EDTA (X TBE) and had been run at Vcm for hr.DSBs had been similarly detected on Southern blots, but had been digested with HindIII alone as previously described (Goldfarb and Lichten,), and electrophoresis buffer was supplemented with mM MgCl.Gels had been transferred to membranes and hybridized with radioactive probe as described (Allers and Lichten, a, b), and had been imaged and quantified making use of a Fuji FLA phosphorimager and ImageGauge .computer software.HindIIIVDE gel blots have been probed with ARG sequences from to nt relative to ARG coding sequences (Probe , Figure).To right for the low degree of uncut VDE web-sites present in all VDE digests (see Figure), NCO frequencies measured from these digests had been adjusted by subtracting the frequency of apparent NCOs in hr samples.HindIII gel blots had been probed with sequences from the DED gene ( to nt relative to DED coding sequence), which can be quickly upstream of ARG (Probe , Figure).Digests of saeD strains (mechanism of action Figure figure supplement) were probed with nt of pBR.Chromatin immunoprecipitation and quantitative PCRCells were formaldehydefixed by adding ml of a .formaldehyde resolution (Sigma) to ml of meiotic cultures, incubating for min at space temperature, and quenched by the addition of glycine to mM.Cells have been harvested by centrifugation, resuspended in ml lysis buffer (StrahlBolsinger et al) except with mgml Bacitracin and full protease inhibitor cocktail (a single tablet ml, Roche) as protease inhibitors, and cells have been lysed at by means of cycles of vortexing on a.