RAD6 gene is involved in heat shock induction of bleomycin resistance in Saccharomyces cerevisiae.

Cells react to environmental and endogenous challenges such as high temperature, reactive oxygen species, DNA damage, and nutrient starvation by activating several defense mechanisms known as stress responses. An important feature is the overlap between different stress responses that contributes at least in part to the phenomenon of cross-protection. We previously demonstrated that pretreatment with a heat shock (HS) induces resistance to the lethal and mutagenic effects of the antineoplastic drug Bleomycin (BLM) in wild-type Saccharomyces cerevisiae. At the DNA level, the HS resulted in more efficient repair of BLM-induced DNA damage. In the present study, we have investigated the mechanisms involved in this HS-induced BLM resistance. Since the RAD6 gene is involved in the ubiquitin system and DNA repair, we analyzed the effects of HS on the lethality of BLM in a rad6Delta (ubc2) mutant strain of S. cerevisiae. The rad6Delta mutant was more sensitive to the lethal effects of BLM than wild-type yeast and HS had no effect on the lethality of BLM in the mutant. Analysis of cell proliferation kinetics indicated that the HS-induced cell cycle delay observed in the wild-type yeast was absent in the rad6Delta mutant strain. BLM treatment impaired mutant cell proliferation, and HS had no effect on the delayed cell kinetics of the mutant. In addition, pulsed-field electrophoresis of chromosomes damaged by BLM indicated that there was very little recovery from damage in the mutant after 24 hr of incubation in BLM-free nutrient medium, and that HS had little effect on the recovery. These data indicate that the RAD6 gene is involved in the HS-induced BLM resistance observed in the isogenic wild-type strain.

HDF1 and RAD17 genes are involved in DNA double-strand break repair in stationary phase Saccharomyces cerevisiae.

DNA repair, checkpoint pathways and protection mechanisms against different types of perturbations are critical factors for the prevention of genomic instability. The aim of the present work was to analyze the roles of RAD17 and HDF1 gene products during the late stationary phase, in haploid and diploid yeast cells upon gamma irradiation. The checkpoint protein, Rad17, is a component of a PCNA-like complex-the Rad17/Mec3/Ddc1 clamp-acting as a damage sensor; this protein is also involved in double-strand break (DBS) repair in cycling cells. The HDF1 gene product is a key component of the non-homologous end-joining pathway (NHEJ). Diploid and haploid rad17Delta/rad17Delta, and hdf1Delta Saccharomyces cerevisiae mutant strains and corresponding isogenic wild types were used in the present study. Yeast cells were grown in standard liquid nutrient medium, and maintained at 30 degrees C for 21 days in the stationary phase, without added nutrients. Cell samples were irradiated with (60)Co gamma rays at 5 Gy/s, 50 Gy

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.

RAD 6 gene is involved in heat shock induction of bleomycin resistance in saccharomyces cerevisiae

Cells react to environmental and endogenous challenges such as high tempertature, reactive oxygen species, DNA damage, and nutrient starvation by activating several defense mechanisms known as stress responses. An important feature is the overlap between different stress responses that contributes at least in part to the phenomenon of cross-protection. We previously demonstrated that pretreatment with a heat shock (HS) induces resistance to the lethal and mutagenic effects of the antineoplastic drug Bleomycin (BLM) in wild-type Saccharomyces cerevisiae. At the DNA level, the HS resulted in more efficient repair of BLM-induced DNA damage. In the present study, we have investigated the mechanisms involved in this HS-induced BLM resistance. Since the RAD6 gene is involved the ubiquitin system and DNA repair, we analyzed the effects of HS on the lethality of BLM in a rad6 (ubc2) mutant strain of S. cerevisiae. The rad6 mutant was more sensitive to the lethal effects of BLM than wild-type yeast and HS had no effect on the lethality of BLM in the mujtant. Analysis of cell proliferation kinetics indicated that the HS-induced cell cycle delay observed in the wild-type yeast was absent in the rad6 mutant strain. BLM treatment impaired mutant cell proliferation, and HS had no effect on the delayed cell kinetics of the mutant. In addition, pulsed-fiel electrophoresis of chromosomes damaged by BLM indicated that there was ery little recovery from damage in the mutant after 24 hr of incubation in BLM-free nutrient medium, and that HS had little effect on the recovery. These data indicate that the RAD6 gene is involved in the HS-induced BLM resistance observed in the isogenic wild.type strain