Abstract
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.
Highlights
Arsenic is a toxic element ubiquitously present in the environment
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
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
Summary
Arsenic is a toxic element ubiquitously present in the environment. Carcinogenic properties of arsenic have been known for a long time and chronic exposure to arsenic in humans has been implicated in numerous types of cancer, including skin, lung, liver, kidney and bladder cancer [1]. Since exposure of millions of people to high doses of arsenic in drinking water constitutes a serious health problem [4] and because of increasing use of arsenic as therapeutic agent [5], it is of great importance to elucidate the mechanisms of arsenic toxicity and tolerance. Several mechanisms have been proposed to explain carcinogenicity of arsenic, including increased formation of reactive oxygen species (ROS) causing oxidative DNA damage such as single-strand breaks (SSBs) that can be processed to double-strand breaks (DSBs) during replication, inhibition of DNA repair and enhancing mutagenicity and carcinogenicity of other factors, like UV light, global changes in DNA methylation and histone modifications and spindle disruption [6]. In human cell lines exposed to arsenic an accumulation of oxidative DNA damage in the form of 8-hydroxy-29-deoxyguanosine (8-OHdG) has been shown, which is reversed by addition of antioxidants [7]
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