Identification of novel genes involved in DNA damage response by screening a genome-wide Schizosaccharomyces pombe deletion library

Abstract

BackgroundDNA damage response (DDR) plays pivotal roles in maintaining genome integrity and stability. An effective DDR requires the involvement of hundreds of genes that compose a complicated network. Because DDR is highly conserved in evolution, studies in lower eukaryotes can provide valuable information to elucidate the mechanism in higher organisms. Fission yeast (Schizosaccharomyces pombe) has emerged as an excellent model for DDR research in recent years. To identify novel genes involved in DDR, we screened a genome-wide S. pombe haploid deletion library against six different DNA damage reagents. The library covered 90.5% of the nonessential genes of S. pombe.ResultsWe have identified 52 genes that were actively involved in DDR. Among the 52 genes, 20 genes were linked to DDR for the first time. Flow cytometry analysis of the repair defective mutants revealed that most of them exhibited a defect in cell cycle progression, and some caused genome instability. Microarray analysis and genetic complementation assays were carried out to characterize 6 of the novel DDR genes in more detail. Data suggested that SPBC2A9.02 and SPAC27D7.08c were required for efficient DNA replication initiation because they interacted genetically with DNA replication initiation proteins Abp1 and Abp2. In addition, deletion of sgf73+, meu29+, sec65+ or pab1+ caused improper cytokinesis and DNA re-replication, which contributed to the diploidization in the mutants.ConclusionsA genome-wide screen of genes involved in DDR emphasized the key role of cell cycle control in the DDR network. Characterization of novel genes identified in the screen helps to elucidate the mechanism of the DDR network and provides valuable clues for understanding genome stability in higher eukaryotes.

Glycan Alteration Imparts Cellular Resistance to a Membrane-Lytic Anticancer Peptide

“…Thus, resistance may be a consequence of mutant cell aggregation, rather than inhibition of the peptide’s ability to electrostatically engage the surface glycans of individual yeast cells. We tested this possibility by evaluating the SVS-1 sensitivity of different deletion mutants selected for their known potential to readily flocculate (Dekker et al, 2004; Drewes and Nurse, 2003; Kim et al, 2001; Linder et al, 2008; Pan et al, 2012; Prevorovsky et al, 2009; Watson and Davey, 1998). All of these mutants were sensitive to the SVS-1 peptide (Figure S3B), indicating that flocculation propensity alone does not confer resistance to yeast.…”

Septin ring assembly is regulated by Spt20, a structural subunit of SAGA complex

“…In a genome-wide screen of genes involved in the DNA damage response, we previously found that spt20D cells exhibited a peak of 4C DNA content in a flow cytometry analysis (Pan et al, 2012). Duplication of DNA content could be caused by DNA rereplication, defect in cytokinesis or cell separation.…”

Caffeine stabilises fission yeast Wee1 in a Rad24-dependent manner but attenuates its expression in response to DNA damage identifying a putative role for TORC1 in mediating its effects on cell cycle progression

“…Caffeine and torin1 clearly increased sensitivity to phleomycin in gaf1Δ mutants, suggesting this effect occurs independently of Gaf1. Rapamycin could enhance sensitivity to the tco89Δ mutant which displays enhanced sensitivity to the drug and other genotoxins (Nakashima et al, 2012, Pan et al, 2012. This activity was not associated with DNA damage checkpoint override.…”