NGS-based analysis of mutation signatures during the translesion synthesis

描述(由申请人提供)：跨损伤脱氧核糖核酸聚合酶，POLζ，REV1和POLη是在脱氧核糖核酸损伤后存活和在幸存者中引入突变的关键酶。由于许多抗癌药物都是DNA损伤剂，TLS聚合酶的活性很可能决定了药物的细胞毒性和治疗所致突变的风险。这项研究的长期目标是阐明特定聚合酶在特定损伤的DNA合成过程中引入的突变类型和频率，以及调节突变的机制。这些信息对于评估致癌物的风险和潜在地控制此类药物的致癌作用至关重要。本项目旨在利用下一代测序技术，通过对酿酒酵母的POLζ、POLη和REV1进行体外培养，确定其准确的突变类型和突变特征。将检查几种DNA损伤，包括烷基化、氧化、胸腺嘧啶二聚体和碱性位点。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反应中需要独特的特征，因为聚合酶应该在反应过程中切换。将剖析Rev1在突变特征中的作用，以阐明催化域和蛋白质相互作用域的作用。该项目还将调查诱变的潜在调控机制。具体的焦点是单一的增殖细胞核抗原和POLζ的非催化亚单位(Rev7，Pol31和Pol32)。这些蛋白质因子对体外TLS反应不是必需的，但可能会影响TLS产物的突变特征。本项目这一部分的目标是确定受调控的亚基，并确定它们如何影响突变签名。该项目还将研究本身不会造成损害的DNA嵌入物诱导的突变。人们对非破坏性诱变剂的诱变机制知之甚少。假设TLS聚合酶的保真度被嵌入剂降低，突变特征将在几个嵌入剂存在的情况下确定。结果：该项目将提供与特定DNA损伤、TLS聚合酶及其相关蛋白相关的突变风险的定量数据。这将改善对突变的机械性洞察。所获得的信息可用于癌症预防和控制。此外，该项目还将开发一种新的方法，可用于评估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.