tivity to radiation. A haploid deletion library of S. pombe was created by Korea Research Institute of Biotechnology and Bioscience and supplied by Bioneer Corporation. This commer cial library facilitates the genome wide screen in fission yeast. By using http://www.selleckchem.com/products/Imatinib-Mesylate.html this library, colleagues identified 229 genes relevant to DDR, among which 23 genes were previously uncharacterized. Following, an upgraded library was applied to investigate the global fitness of deletions after different kinds of DNA damage by barcode sequencing. Both studies made impressive progress to gain a bet ter understanding of DDR. However, the deletion libraries applied in these studies only covered around 70% of non essential S. pombe genes. In this sense, screening a deletion library with a higher coverage of genes seemed worthwhile in order to build a more comprehensive DDR network.
In this study, we screened a Inhibitors,Modulators,Libraries S. pombe haploid deletion library, containing 3,235 deletions, against six different DNA damage reagents. The Inhibitors,Modulators,Libraries library represented approxi mately 90. 5% of non essential genes in the genome. 52 genes were identified to be closely related with DDR, 20 of which were reported for the first time. We characterized six novel DDR genes by flow cytometry and microarray analysis. Data suggest these genes might function in DNA replication and cytokinesis, providing a basis for further characterization of their roles in DDR. Results Genome wide screen of DNA damage sensitive mutants Six chemical reagents that can cause different kinds of DNA damage were chosen for the screen.
Hydroxyurea inhibits ribonucleotide reductase, depletes nucleotides pool and thus leads to an S phase arrest. Bleomycin, a mimetic of gamma irradiation, causes double strand breaks. Methyl Inhibitors,Modulators,Libraries methanesulfonate, an alkylating agent, primarily methylates DNA on N7 Inhibitors,Modulators,Libraries deoxy guanine and N3 deoxyadenine, leading to DNA synthesis defects. Camptothecin locks topoisomerase I covalently onto the DNA and thus causes strand breaks during S phase. Ultraviolent radiation results in an abnormal covalent bond between adjacent pyrimidine bases. Thiabendazole depolymerizes the micro tubule and was used to check the integrity of the spindle checkpoint. Before the screen was performed, the growth of WT cells with different concentrations of DNA damaging agents were monitored. The highest concentra tion that did not affect the growth of WT cells was chosen for large scale screen.
By using this concentration, it was easier to compare the growth with WT cells and to pick the sensitive mutants. The screen was carried out in three rounds. Entinostat First, 3,235 deletions were exposed to each DNA damage reagent in 96 well microtiter plates. 630 mutants showing sensitivities to at least one reagent were picked to create a sub library. In the second round, mutants from the sub library were grown in test tubes U0126 ERK to repeat the sensitivity assays, and 322 sensitive deletions were obtained. In the last round of the screen, 322 deletions were subjected to spot assa