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.

Antioxidant activity of a botanical extract preparation of Ilex paraguariensis: prevention of DNA double-strand breaks in Saccharomyces cerevisiae and human low-density lipoprotein oxidation.

We analyzed the antioxidant properties of Ilex paraguariensis infusion (Ip) popularly known as mate (mä'ta), by using two experimental models: the induction of DNA double-strand breaks (DSB) by hydrogen peroxide (H(2)O(2)) and lethality in Saccharomyces cerevisiae, as well as peroxide and lipoxygenase-induced human low-density lipoprotein (LDL) oxidation. Diploid yeast cells were exposed to different concentrations of H(2)O(2) (5-10 mmol/L) in the absence or presence of Ip infusion (10(-1) v/v) or alpha-tocopherol (10(-2) mol/L). Both mate infusion and alpha-tocopherol significantly decreased the dose dependent DSB number, and the lethality induced by H(2)O(2). Peroxynitrite and lipoxygenase-induced human LDL oxidation are inhibited by Ip extracts in a potent, dose-dependent fashion. Dilutions of 5 x 10(-3) v/v provide 50% +/- 10% inhibition. Finally, Ip extracts are potent direct quenchers of the free radical 1,1-diphenyl-2-picrylhydrazyl. Dilutions of 2 x 10(-2) v/v produced quenching of more than 30%, which was comparable to that obtained with 0.5-1 mmol/L alpha-tocopherol or the quercetin aglycone, respectively. For comparison, total polyphenol content of Ip, green, and black tea (Camelia sinensis) were 6.5 +/- 0.8; 1.8 +/- 0.5; and 1.13 +/- 0.3 mmol of quercetin equivalents per liter, respectively. Their respective free radical quenching activities at dilutions of 1 x 10(-1) v/v were 75% +/- 5%; 35% +/- 5%; and 2% +/- 5%. Ip is thus a rich source of polyphenols and has antioxidant properties comparable to those of green tea which merit further in vivo intervention and cross-sectional studies.