NGS-based analysis of mutation signatures during the translesion synthesis

描述（由申请人提供）：转录DNA聚合酶、Pol η、Rev 1和Polη是DNA损伤后存活和在存活者中引入突变的关键酶。由于许多抗癌药物是DNA损伤剂，TLS聚合酶的活性可能决定了药物的细胞毒性和治疗诱变的风险。本研究的长期目标是阐明特定聚合酶在DNA合成过程中引入突变的类型和频率，以及调控突变的机制。这些信息对于评估致癌物的风险并可能控制此类药物的致癌作用至关重要。 本项目的具体目的是通过S的Pol、Polη和Rev1确定体外TLS期间的精确突变类型和速率（突变特征）。酿酒酵母，通过使用下一代测序（NGS）技术。将检查几种DNA损伤，包括烷基化、氧化、胸腺嘧啶二聚体和脱碱基位点。 该项目将确定特定于聚合酶类型和损伤类型的突变特征。此外，在多聚合酶TLS反应中预期独特的签名，因为聚合酶应该在反应期间转换。Rev1在突变特征中的作用将被解剖，以澄清催化和蛋白质相互作用结构域的作用。 本项目还将研究突变的潜在调控机制。具体重点是monoubiquitinated PCNA和非催化亚基的Poll（Rev7，Pol31和Pol32）。这些蛋白因子对于体外TLS反应不是必需的，但可能影响TLS产物中的突变特征。这部分项目的目标是识别调控亚基，并确定它们如何影响突变特征。 本项目还将研究由本身不引起损伤的DNA嵌入剂引起的诱变。非损伤性诱变剂的致突变机制目前还知之甚少。假设TLS聚合酶的结晶度被嵌入剂降低，突变特征将在几种嵌入剂的存在下确定。结果：该项目将提供关于伴随确定的DNA损伤、TLS聚合酶及其相关蛋白的突变风险的定量数据。这将提高对诱变机理的认识。获得的信息可用于癌症预防和控制。此外，本项目还将开发新的方法，可用于评价许多其他化学品的遗传毒性。

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.