The Cellular Roles of the Translesion Polymerases Eta, REV1 and Zeta in Bypass and Repair of DNA Damage Induced by Anti-Cancer Agents.

Abstract

Translesion DNA synthesis (TLS) is a process whereby specialized DNA polymerases are recruited to bypass DNA lesions that would otherwise stall high-fidelity polymerases. TLS may be a mechanism by which cancer cells resist the cytotoxic actions of chemotherapeutic agents that damage DNA. Here, I addressed if the translesion polymerases eta, REV1 and zeta have a cellular role in protecting against cisplatin-induced cytotoxicity. HeLa cells depleted of polymerase eta, REV1 or polymerase zeta individually were found to display phenotypes suggestive of the inability to bypass cisplatin adducts. In addition, the E3 ubiquitin ligase RAD18 and the Fanconi anemia core complex were found to be crucial for regulating TLS during bypass of cisplatin adducts. Together, the findings support a model where replicative bypass of cisplatin intrastrand cross-links requires cooperation of multiple translesion polymerases in human cells, and is regulated by both PCNA monoubiquitination via RAD18 and the Fanconi anemia core complex. Recently, translesion DNA polymerases have been implicated in the repair of DNA damage induced by chemotherapeutic agents. I addressed if the translesion polymerases eta, REV1 and zeta were essential for efficient repair of DNA interstrand cross-links (ICLs) induced by mitomycin C and DNA double strand breaks (DSBs) caused by exposure to ionizing radiation (IR). Depletion of REV1 or polymerase zeta in HeLa cells, but not polymerase eta or RAD18, resulted in a loss in clonogenic survival following exposure to both mitomycin C and IR. I demonstrated that the loss in survival following mitomycin treatment is likely due to inefficient repair of ICLs in cells depleted of REV1 or polymerase zeta by measuring the resolution of DSBs, an intermediate of ICL repair. Furthermore, I showed that REV1 and polymerase zeta were required for the timely repair of DSBs induced by ionizing radiation. REV1 and polymerase zeta depleted HeLa cells exhibited a relatively mild but significant defect in homologous recombination, an essential DNA repair pathway important for resolving DSBs. Overall, the results suggest both REV1 and polymerase zeta play an active role in protection against mitomycin C or ionizing radiation-induced cytotoxicity, most likely by directly repairing DNA damage caused by these agents.