Impacts of arsenic on Rad18 and translesion synthesis.

Arsenite interferes with DNA repair protein function resulting in the retention of UV-induced DNA damage. Accumulated DNA damage promotes replication stress which is bypassed by DNA damage tolerance pathways such as translesion synthesis (TLS). Rad18 is an essential factor in initiating TLS through PCNA monoubiquitination and contains two functionally and structurally distinct zinc fingers that are potential targets for arsenite binding. Arsenite treatment displaced zinc from endogenous Rad18 protein and mass spectrometry analysis revealed arsenite binding to both the Rad18 RING finger and UBZ domains. Consequently, arsenite inhibited Rad18 RING finger dependent PCNA monoubiquitination and polymerase eta recruitment to DNA damage in UV exposed keratinocytes, both of which enhance the bypass of cyclobutane pyrimidine dimers during replication. Further analysis demonstrated multiple effects of arsenite, including the reduction in nuclear localization and UV-induced chromatin recruitment of Rad18 and its binding partner Rad6, which may also negatively impact TLS initiation. Arsenite and Rad18 knockdown in UV exposed keratinocytes significantly increased markers of replication stress and DNA strand breaks to a similar degree, suggesting arsenite mediates its effects through Rad18. Comet assay analysis confirmed an increase in both UV-induced single-stranded DNA and DNA double-strand breaks in arsenite treated keratinocytes compared to UV alone. Altogether, this study supports a mechanism by which arsenite inhibits TLS through the altered activity and regulation of Rad18. Arsenite elevated the levels of UV-induced replication stress and consequently, single-stranded DNA gaps and DNA double-strand breaks. These potentially mutagenic outcomes support a role for TLS in the cocarcinogenicity of arsenite.
Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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