Oxidative Stress and Replication-Independent DNA Breakage Induced by Arsenic in Saccharomyces cerevisiae

摘要

Arsenic is a well-established human carcinogen of poorly understood mechanism of genotoxicity. It is generally accepted that arsenic acts indirectly by generating oxidative DNA damage that can be converted to replication-dependent DNA double-strand breaks (DSBs), as well as by interfering with DNA repair pathways and DNA methylation. Here we show that in budding yeast arsenic also causes replication and transcription-independent DSBs in all phases of the cell cycle, suggesting a direct genotoxic mode of arsenic action. This is accompanied by DNA damage checkpoint activation resulting in cell cycle delays in S and G2/M phases in wild type cells. In G1 phase, arsenic activates DNA damage response only in the absence of the Yku70–Yku80 complex which normally binds to DNA ends and inhibits resection of DSBs. This strongly indicates that DSBs are produced by arsenic in G1 but DNA ends are protected by Yku70–Yku80 and thus invisible for the checkpoint response. Arsenic-induced DSBs are processed by homologous recombination (HR), as shown by Rfa1 and Rad52 nuclear foci formation and requirement of HR proteins for cell survival during arsenic exposure. We show further that arsenic greatly sensitizes yeast to phleomycin as simultaneous treatment results in profound accumulation of DSBs. Importantly, we observed a similar response in fission yeast Schizosaccharomyces pombe , suggesting that the mechanisms of As(III) genotoxicity may be conserved in other organisms. Author Summary Arsenic is a highly toxic compound which causes several types of cancer in humans. However, precise mechanisms of arsenic carcinogenesis remain elusive and are still a matter of debate. For example, the oxidative stress theory of arsenic proposes that arsenic generates reactive oxygen species producing oxidative DNA damage that can be converted to DNA double-strand breaks (DSBs) during replication. Using budding yeast as a model organism, we show that arsenic is able to induce DSBs in the absence of transcription, replication and pronounced oxidative stress. Importantly, we also demonstrate that arsenic greatly enhances cytotoxic activity of antitumor drug phleomycin, as evidenced by increased sensitivity and DNA fragmentation visible upon co-treatment. Our work suggests that arsenic acts as a direct inducer of DNA breaks and could be potentially used with other anticancer drugs, like phleomycin-related bleomycin, as a new combinatory therapy to treat cancers that poorly respond to these drugs. Additionally, since in many countries millions of people are exposed to high doses of arsenic in drinking water, we believe that our findings about genotoxicity of arsenic are important not only to geneticists but also to the general public.