Roles of Saccharomyces cerevisiae RAD17 and CHK1 checkpoint genes in the repair of double-strand breaks in cycling cells.
Checkpoints are components of signalling pathways involved in genome stability. We analysed the putative dual functions of Rad17 and Chk1 as checkpoints and in DNA repair using mutant strains of Saccharomyces cerevisiae. Logarithmic populations of the diploid checkpoint-deficient mutants, chk1Delta/chk1Delta and rad17Delta/rad17Delta, and an isogenic wild-type strain were exposed to the radiomimetic agent bleomycin (BLM). DNA double-strand breaks (DSBs) determined by pulsed-field electrophoresis, surviving fractions, and proliferation kinetics were measured immediately after treatments or after incubation in nutrient medium in the presence or absence of cycloheximide (CHX). The DSBs induced by BLM were reduced in the wild-type strain as a function of incubation time after treatment, with chromosomal repair inhibited by CHX. rad17Delta/rad17Delta cells exposed to low BLM concentrations showed no DSB repair, low survival, and CHX had no effect. Conversely, rad17Delta/rad17Delta cells exposed to high BLM concentrations showed DSB repair inhibited by CHX. chk1Delta/chk1Delta cells showed DSB repair, and CHX had no effect; these cells displayed the lowest survival following high BLM concentrations. Present results indicate that Rad17 is essential for inducible DSB repair after low BLM-concentrations (low levels of oxidative damage). The observations in the chk1Delta/chk1Delta mutant strain suggest that constitutive nonhomologous end-joining is involved in the repair of BLM-induced DSBs. The differential expression of DNA repair and survival in checkpoint mutants as compared to wild-type cells suggests the presence of a regulatory switch-network that controls and channels DSB repair to alternative pathways, depending on the magnitude of the DNA damage and genetic background.
Bracesco
Nelson
N
Lab. Radiobiología, Departamento Biofísica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay.
Candreva
Ema C
EC
Keszenman
Deborah
D
Sánchez
Ana G
AG
Soria
Sandra
S
Dell
Mercedes
M
Siede
Wolfram
W
Nunes
Elia
E
D002453:Cell Cycle / Q000502:physiology / Q000528:radiation effects; D018797:Cell Cycle Proteins / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D004249:DNA Damage / Q000502:physiology; D004260:DNA Repair / Q000502:physiology* / Q000528:radiation effects; D004271:DNA, Fungal / Q000502:physiology* / Q000528:radiation effects*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018816:Genes, cdc / Q000502:physiology / Q000528:radiation effects; D009687:Nuclear Proteins / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000502:physiology* / Q000528:radiation effects; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C501395:RAD17 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; D000071877:Checkpoint Kinase 1