RESUMEN
Antimony is a toxic metalloid with poorly understood mechanisms of toxicity and uncertain carcinogenic properties. By using a combination of genetic, biochemical and DNA damage assays, we investigated the genotoxic potential of trivalent antimony in the model organism Saccharomyces cerevisiae. We found that low doses of Sb(III) generate various forms of DNA damage including replication and topoisomerase I-dependent DNA lesions as well as oxidative stress and replication-independent DNA breaks accompanied by activation of DNA damage checkpoints and formation of recombination repair centers. At higher concentrations of Sb(III), moderately increased oxidative DNA damage is also observed. Consistently, base excision, DNA damage tolerance and homologous recombination repair pathways contribute to Sb(III) tolerance. In addition, we provided evidence suggesting that Sb(III) causes telomere dysfunction. Finally, we showed that Sb(III) negatively effects repair of double-strand DNA breaks and distorts actin and microtubule cytoskeleton. In sum, our results indicate that Sb(III) exhibits a significant genotoxic activity in budding yeast.
Asunto(s)
Antimonio/toxicidad , Daño del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , ADN/metabolismo , Roturas del ADN de Doble Cadena/efectos de los fármacos , Daño del ADN/genética , Reparación del ADN/efectos de los fármacos , ADN-Topoisomerasas de Tipo I/metabolismo , Estrés Oxidativo/genética , Recombinación Genética/efectos de los fármacos , Recombinación Genética/genética , Reparación del ADN por Recombinación/efectos de los fármacos , Reparación del ADN por Recombinación/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Telómero/metabolismoRESUMEN
DNA damage tolerance and homologous recombination pathways function to bypass replication-blocking lesions and ensure completion of DNA replication. However, inappropriate activation of these pathways may lead to increased mutagenesis or formation of deleterious recombination intermediates, often leading to cell death or cancer formation in higher organisms. Post-translational modifications of PCNA regulate the choice of repair pathways at replication forks. Its monoubiquitination favors translesion synthesis, while polyubiquitination stimulates template switching. Srs2 helicase binds to small ubiquitin-related modifier (SUMO)-modified PCNA to suppress a subset of Rad51-dependent homologous recombination. Conversely, SUMOylation of Srs2 attenuates its interaction with PCNA Sgs1 helicase and Mus81 endonuclease are crucial for disentanglement of repair intermediates at the replication fork. Deletion of both genes is lethal and can be rescued by inactivation of Rad51-dependent homologous recombination. Here we show that Saccharomyces cerevisiae Uls1, a member of the Swi2/Snf2 family of ATPases and a SUMO-targeted ubiquitin ligase, physically interacts with both PCNA and Srs2, and promotes Srs2 binding to PCNA by downregulating Srs2-SUMO levels at replication forks. We also identify deletion of ULS1 as a suppressor of mus81Δ sgs1Δ synthetic lethality and hypothesize that uls1Δ mutation results in a partial inactivation of the homologous recombination pathway, detrimental in cells devoid of both Sgs1 and Mus81 We thus propose that Uls1 contributes to the pathway where intermediates generated at replication forks are dismantled by Srs2 bound to SUMO-PCNA. Upon ULS1 deletion, accumulating Srs2-SUMO-unable to bind PCNA-takes part in an alternative PCNA-independent recombination repair salvage pathway(s).
