Effects of bleomycin on growth kinetics and survival of Saccharomyces cerevisiae: a model of repair pathways

Abstract

In order to analyze the roles of some repair genes in the processing of bleomycin-induced DNA damage and, especially, the interrelationships among the involved repair pathways, we investigated the potentially lethal effect of bleomycin on radiosensitive mutants of Saccharomyces cerevisiae defective in recombination, excision, and R4D6-dependent DNA repair. Using single, double, and triple rad mutants, we analyzed growth kinetics and survival curves as a function of bleomycin concentration. Our results indicate that genes belonging to the three epistasis groups interact in the repair of bleomycin-induced DNA damage to different degrees depending on the concentration of bleomycin. The most important mechanisms involved are recombination and postreplication repair. The initial action of a potentially inducible excision repair gene could provide intermediate substrates for the R4D6-and RAD52-dependent repair processes. Interaction between R4D6 and RADS2 genes was epistatic for low bleomycin concentrations. RA1D3 and RAD52 genes act independently in processing DNA damage induced by high concentrations of bleomycin. The synergistic interaction observed at high concentrations in the triple mutant rad2-6 rad6-1 rad52-1 indicates partial independence of the involved repair pathways, with possible common substrates. On the basis of the present results, we propose a heuristic model of bleomycin-induced DNA damage repair.Bleomycin (BLM) is a radiomimetic glycopeptide antibiotic used extensively in tumor therapy (32,48). This drug binds to DNA and through a free-radical-based mechanism produces DNA base loss and single-and double-strand breaks (43,44). In different lines of eukaryotic cells, BLM produces inhibition of DNA synthesis (10) as well as cell cycle G2 delay (19,38). With phages, bacteria, and yeast cells, it was demonstrated that this drug is recombinogenic and mutagenic (11,23,39,42). Analysis of BLM-induced mutagenesis in repackaged lambda phage suggests SOS repair dependence (33).BLM-induced DNA damage in prokaryotic and eukaryotic cells is partially reparable (3,22,27,40). For human fibroblasts, a long-patch mode of excision repair of BLM-induced damage was recently described (4, 5). Using haploid and diploid radiosensitive strains of Saccharomyces cerevisiae, Moore (22) showed that all rad single mutants sensitive to BLM tested, with the exception of rad1S, are sensitive to X rays. Likewise, BLM-sensitive (blm) mutants exhibit crosssensitivity to ionizing radiation and hydrogen peroxide (26). These results suggest that some aspects of the repair of BLM and ionizing radiation DNA damage in yeast cells may be similar. Furthermore, DNA ligase (the product of the CDC9 gene) is required in the overall process of restoring full-size DNA molecules from DNA fragments produced by BLM (24, 25).We have recently shown that BLM induces at least one error-prone type of repair in S. cerevisiae and that the PS04 (XS9) gene acts as a mutation-triggering factor (39). Furthermore, the combination of UV light and BLM produces di...

A Genome-Wide Screen in Saccharomyces cerevisiae Reveals Altered Transport As a Mechanism of Resistance to the Anticancer Drug Bleomycin

“…Alternatively, the 2-fold increase in F-BLM uptake may lead to more toxic DNA lesions. In fact, it is known that distinct DNA lesions are generated in yeast cells in a manner that depends on the BLM intracellular concentration (48). Nonetheless, the consequences of altering Agp2 levels appear to be specific for BLM, because either Agp2-overproducing or -null strains show parental resistance to several other diverse genotoxic agents including MMS, 4-NQO, cisplatin, and camptothecin ( Fig.…”

Requirement for End-Joining and Checkpoint Functions, but NotRAD52-Mediated Recombination, after EcoRI Endonuclease Cleavage of Saccharomyces cerevisiaeDNA

“…It is therefore likely that such ends require extensive processing and/or recombinational repair to restore the intact, unmutagenized strand. RAD52 is also required for the repair of DSBs containing modified termini such as those generated by various chemical clastogens, e.g., bleomycin (29,46). Furthermore, we have recently observed that high-level expression of PvuII, which generates blunt termini, produces much greater killing in rad52 cells than in Rad ϩ strains (36).…”

A Double-Strand Break Repair Component Is Essential for S Phase Completion in Fission Yeast Cell Cycling

“…Various organisms including the fission yeast Schizosaccharomyces pombe and mammals contain counterparts of these factors (Bezzubova et al, 1993;Muris et al, 1993Muris et al, , 1994Ostermann et al, 1993;Shinohara et al, 1993;Kanaar et al, 1996;Albala et al, 1997). In budding and fission yeast, these factors are dispensable for viability although required for mating type switching and repair of chemically or physically induced double-strand breaks (McKee and Lawrence, 1980;Borts et al, 1986;Kezenman et al, 1992;Schlake et al, 1993). However, in higher eukaryotes, some of these factors are indispensable for viability because of requirement for repair of the double-strand breaks that are spontaneously pro-duced during chromosomal replication at least in some cells (Lim and Hasty, 1996;Tsuzuki et al, 1996).…”