RESUMO
Yeast mutants, snm1 (pso2-1), rev3 (pso1-1), and rad51, which display significant sensitivity to interstrand crosslinks (ICLs) have low relative sensitivity to other DNA damaging agents. SNM1, REV3, and RAD51 were disrupted in the same haploid strain, singly and in combination. The double mutants, snm1 Delta rev3 Delta, snm1 Delta rad51 Delta and rev3 Delta rad51 Delta were all more sensitive to ICLs than any of the single mutants, indicating that they are in separate epistasis groups for survival. A triple mutant displayed greater sensitivity to ICLs than any of the double mutants, with one ICL per genome being lethal. Therefore, Saccharomyces cerevisiae appears to have three separate ICL repair pathways, but no more. S-phase delay was not observed after ICL damage introduced by cisplatin (CDDP) or 8-methoxypsoralen (8-MOP) during the G1-phase, in any of the above mutants, or in an isogenic rad14 Delta mutant deficient in nucleotide excision repair. However, the psoralen analog angelicin (monoadduct damage) induced a significant S-phase delay in the rad14 Delta mutant. Thus, normal S-phase in the presence of ICLs does not seem to be due to rapid excision repair. The results also indicate that monoadduct formation by CDDP or 8-MOP at the doses used is not sufficient to delay S-phase in the rad14 Delta mutant. While the sensitivity of a rev3 Delta mutant indicates Pol zeta is needed for optimal ICL repair, isogenic cells deficient in Pol eta (rad30 Delta cells) were not significantly more sensitive to ICL agents than wild-type cells, and have no S-phase delay.
Assuntos
Reparo do DNA/fisiologia , DNA Fúngico/efeitos dos fármacos , Proteínas de Ligação a DNA/fisiologia , Proteínas Fúngicas/fisiologia , Proteínas Nucleares/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Cisplatino/farmacologia , Reagentes de Ligações Cruzadas/farmacologia , Dano ao DNA , Reparo do DNA/genética , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/fisiologia , Endodesoxirribonucleases , Epistasia Genética , Proteínas Fúngicas/genética , Furocumarinas/farmacologia , Metoxaleno/farmacologia , Proteínas Nucleares/genética , Rad51 Recombinase , Fase S/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The role of Snm1, Rev3 and Rad51 in S-phase after cisplatin (CDDP) DNA treatment has been examined. When isogenic deletion mutants snm1 delta, rev3 delta and rad51 delta were arrested in G1 and treated with doses of CDDP causing significant lethality (<20% survival in the mutant strains), they progressed through S-phase with normal kinetics. The mutants arrested in G2 like wild-type cells, however they did not exit the arrest and reenter the cell cycle. This finding demonstrates that these genes are not required to allow DNA replication in the presence of damage. Therefore, Snm1, Rev3 and Rad51 may act after S to allow repair. At high levels of damage (<40% survival in wild-type cells) S-phase was slowed in a MEC1-dependent fashion. The cross-link incision kinetics of snm1 delta and rev3 delta mutants were also examined; both showed no deficiencies in incision of cross-linked DNA.
Assuntos
Cisplatino/farmacologia , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA , Proteínas Fúngicas/genética , Interfase/genética , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae , Antineoplásicos/farmacologia , Reagentes de Ligações Cruzadas/farmacologia , Dano ao DNA , Reparo do DNA , Replicação do DNA , Endodesoxirribonucleases , Furocumarinas/farmacologia , Fase G2/genética , Deleção de Genes , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Serina-Treonina Quinases , Rad51 Recombinase , Fase S/genética , Saccharomyces cerevisiaeRESUMO
Cisplatin (CDDP) has been used as a DNA cross-linking agent to evaluate whether there is a specific cell cycle checkpoint response to such damage in Saccharomyces cerevisiae (S. cerevisiae). Fluorescent-activated cell sorting (FACS) analysis showed only a G2/M checkpoint, normal exit from G1 and progression through S-phase following alpha-factor arrest and CDDP treatment. Of the checkpoint mutants tested, rad9, rad17 and rad24, did not show increased sensitivity to CDDP compared to isogenic wild-type cells. However, other checkpoint mutants tested (mec1, mec3 and rad53) showed increased sensitivity to CDDP, as did controls with a defect in excision repair (rad1 and rad14) or a defect in recombination (rad51 and rad52). Thus, by survival and cell cycle kinetics, it appears that DNA cross-links do not inhibit entry into S-phase or slow DNA replication and that replication continues after cisplatin treatment in yeast.
