Further phenotypic characterization of pso mutants of Saccharomyces cerevisiae with respect to DNA repair and response to oxidative stress

The sensitivity responses of seven pso mutants of Saccharomyces cerevisiae towards the mutagens N-nitrosodiethylamine (NDEA), 1,2:7,8-diepoxyoctane (DEO), and 8-hydroxyquinoline (8HQ) further substantiated their allocation into two distinct groups: genes PSO1 (allelic to REV3), PSO2 (SNM1), PSO4 (PRP19), and PSO5 (RAD16) constitute one group in that they are involved in repair of damaged DNA or in RNA processing whereas genes PSO6 (ERG3) and PSO7 (COX11) are related to metabolic steps protecting from oxidative stress and thus form a second group, not responsible for DNA repair. PSO3 has not yet been molecularly characterized but its pleiotropic phenotype would allow its integration into either group. The first three PSO genes of the DNA repair group and PSO3, apart from being sensitive to photo-activated psoralens, have another common phenotype: they are also involved in error-prone DNA repair. While all mutants of the DNA repair group and pso3 were sensitive to DEO and NDEA the pso6 mutant revealed WT or near WT resistance to these mutagens. As expected, the repair-proficient pso7-1 and cox11-Delta mutant alleles conferred high sensitivity to NDEA, a chemical known to be metabolized via redox cycling that yields hydroxylamine radicals and reactive oxygen species. All pso mutants exhibited some sensitivity to 8HQ and again pso7-1 and cox11-Delta conferred the highest sensitivity to this drug. Double mutant snm1-Delta cox11-Delta exhibited additivity of 8HQ and NDEA sensitivities of the single mutants, indicating that two different repair/recovery systems are involved in survival. DEO sensitivity of the double mutant was equal or less than that of the single snm1-Delta mutant. In order to determine if there was oxidative damage to nucleotide bases by these drugs we employed an established bacterial test with and without metabolic activation. After S9-mix biotransformation, NDEA and to a lesser extent 8HQ, lead to significantly higher mutagenesis in an Escherichia coli tester strain WP2-IC203 as compared to WP2, whereas DEO-induced mutagenicity remained unchanged

2-Deoxy-D-glucose induced modulation of DNA damage repair, survival, mutagenesis and recombinogenesis in 8-MOP+UVA treated yeast.

Cellular and genomic effects of post-treatment repair modulation by 2-deoxy-D-glucose (2-DG) and yeast extract were studied in 8-MOP + UVA treated cells of Saccharomyces cerevisiae. The type of lesions and their repair in phosphate buffer glucose (PBG) differed with UVA dose. At low UVA dose (1.4 kJ/m2), lesions were sublethal and mutagenic and did not repair by recombinogensis. The fraction of potentially lethal lesions and lesions repaired by recombinogenesis increased with UVA dose. Cellular repair in PBG was largely error-free and was inhibited by 2-DG. Yeast extract enhanced cellular repair and also recombinogensis; 2-DG in presence of yeast extract promoted error-prone repair. Pulsed-field gel electrophoresed chromosomal DNA bands did not show observable alterations immediately after 8-MOP + UVA treatment. On post-treatment incubation in PBG, the intensity ratio (rho n), of each band altered in a biphasic manner showing decrease first, followed by either increase or no change upto 24 hr depending upon UVA exposure dose. Presence of 2-DG in PBG inhibited decrease in rho n in a concentration dependent manner. Yeast extract reduced the time of first phase of DNA repair. 2-DG and yeast extract together reduced the time of first phase of repair and also inhibited the subsequent increase in rho n, which was observed in the case of yeast extract in PBG. It is proposed that (i) 2-DG in PBG inhibits excision of DNA damage and error-free repair; (ii) yeast extract stimulates the error-prone repair associated with cell cycle and recombinogenesis; (iii) 2-DG in presence of yeast extract allows excision of damage but inhibits build up through recombinogenesis inducing instead, cell cycle associated error-prone repair. A simple schematic model has been proposed to explain these events.