As a sole source for histones H3 and H4. Histone H3 point mutations have been created in HIS3-based plasmids (pRS413-H3H4-3F12), transformed into AKY796 and AKY1037, and counter-selected on 5-FOA plates to get strains with H3 mutations. All histone expression plasmids are listed in Supplementary Table S2. To create Rpb9 anchor-away strains, the RPB9 locus was replaced with rpb9-FRB-hphMX expression cassette in strain HHY168 (Euroscarf)38. In Rpb9 anchor-away strains AKY1162 and AKY1190, HIS3-based plasmid with wild form H3 or H3 K9,14,23 R mutant was introduced as a sole supply for H3 and H4 genes. The rad53 strain was constructed by 1st replacing the SML1 gene with kanMX6 (AKY1438) and after that RAD53 was replaced with URA3 to obtain AKY1459 (RAD53 deletion is viable only in sml1 background). yEGFP was fused to C-terminus of Rad52 in its native locus in AKY1551 strain. Plasmid with galactose inducible HO endonucleaseSciEntific RepoRts (2018) eight:2949 DOI:10.1038/s41598-018-21110-Methodswww.nature.com/scientificreports/pGALHO-pRS41255 was a gift from Dr. Jeff Thompson. Strain LS50 with GAL-HO integrated in to the genome was a gift from Dr. Lena Str . This strain was Methoxyfenozide supplier utilised to create AKY1390. Phosphorylated H2A was detected by anti -H2A antibody (ab15083, Abcam). Histone H3 was C-terminally tagged with E2-tag and detected with 5E11 antibody (Icosagen), Rad53 was tagged with Flag-tag and detected with M2 antibody (��-Cyano-4-hydroxycinnamic acid custom synthesis Sigma-Aldrich). For western blot, cell extracts had been ready as described56 and protein samples have been separated on SDS-polyacrylamide gel. For development curve evaluation, exponentially increasing yeast cultures have been inoculated into 10 ml fresh YPD media at density five ?106 cells per ml. Cells were grown further in a shaker at 30 and samples had been collected at indicated time-points in the course of the development. Culture density was measured with Z2 Cell and Particle Counter (Beckman Coulter). For spot test assays, 10-fold serial dilutions of cell suspensions were made and 5 of each and every dilution was spotted onto plates with synthetic total (SC) selective medium. For plasmid shuffling, 1 mg/ml 5-fluoroorotic acid (5-FOA) and indicated concentrations of MMS in SC plates were utilized to test viability of cells. In experiments with Rpb9 anchor-away strains, 1 /ml rapamycin (Cayman Europe) in 0.1 DMSO as a final concentration was added for the cultures (0.1 DMSO was used for controls). Plates had been incubated a minimum of 2 days at 30 . For flow cytometry evaluation of cell cycle, 0.five ml of yeast culture was fixed in ten ml of ice-cold 70 ethanol for no less than 15 min and washed after with 50 mM citric acid. RNA was degraded with RNase A (ten g/ml) in 50 mM citric acid overnight at 37 . DNA was stained with ten?SYBR Green I (Invitrogen) in 50 mM citric acid for 30 min. Cells have been analysed with FACS Aria, cell cycle distribution was analysed with Cyflogic software program.Yeast development assays and flow cytometry.Fluorescence microscopy.For cell morphology evaluation, cells have been fixed with 70 ethanol and stained with four,6-Diamidino-2-Phenylindole (DAPI). Cells have been imaged working with Olympus BX61 microscope at 100?magnification. Rad52-GFP foci had been detected in vivo from live S-G2 cells. For quantification at least one hundred cells from 3 independent experiments were counted. For MMS-induction of Rad52 foci, Rpb9 was depleted from cells for six hours and treated with 0.1 MMS for 1 hour. All photos were collected with cellSens software program and analysed in ImageJ.DSB repair evaluation.For detection of DSB repair in M.
