NGS-based analysis of mutation signatures during the translesion synthesis

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DESCRIPTION (provided by applicant): Translesion DNA polymerases, Polζ, Rev1, and Polη are key enzymes for surviving after DNA damage and for introducing mutations in the survivors. Since many anti-cancer drugs are DNA damaging agents, it is likely that the activities of TLS polymerases determine the cytotoxicity of the drugs and the risk of mutagenesis by the treatment. The long-term goal of this study is to elucidate the types and frequency of mutations introduced by specific polymerase during the DNA synthesis over specific damage, and the mechanisms that regulate the mutagenesis. Such information is crucial to evaluate the risks of carcinogens and potentially control the carcinogenic effects of such drugs. This project specifically aims to determine precise mutation types and rates (mutation signatures) during the in vitro TLS by Polζ, Polη, and Rev1 of S. cerevisiae, by using the next-generation sequencing (NGS) technology. Several DNA damages, including alkylation, oxidation, thymine-dimer, and abasic site will be examined. This project will determine the mutation signatures that are specific to polymerase types and damage-types. Furthermore, unique signatures are expected in multi-polymerase TLS reactions, since polymerase should switch during the reactions. Role of Rev1 in the mutation signatures will be dissected to clarify the roles of catalytic and protein interaction domains. This project will also investigate potential regulatory mechanisms of the mutagenesis. Specific focuses are on monoubiquitinated PCNA and non-catalytic subunits of Polζ (Rev7, Pol31, and Pol32). These protein factors are not essential for TLS reaction in vitro, but may affect the mutation signatures in the TLS products. Goal of this part of the project is to identify the regulatory subunits and to determine how they affect the mutation signatures. This project will also investigate the mutagenesis induced by DNA intercalators that do not induce damage by itself. Little is known about the mechanisms of mutagenesis by the nondamaging mutagens. Hypothesizing that the fidelities of TLS polymerases are decreased by intercalators, mutation signatures will be determined in the presence of several intercalators. Outcome: This project will provide quantitative data about the risk of mutations that are accompanied with defined DNA damages, TLS polymerases and their associating proteins. It will improve mechanistic insights about the mutagenesis. Obtained information can be used for cancer prevention and control. In addition, this project will develop new method that can be used to evaluate the genotoxicity many other chemicals.