低保真性DNA聚合酶Pol kappa N-Clasp结构调控正确跨越BP-dG加合物效应和形成DNA-蛋白交联的机制研究

Translesion DNA synthesis (TLS) is one mode of DNA damage tolerance, which could utilize translesion DNA polymerases to traverse lesions that are blocking replication. TLS can be classified into two categories: error-free and error-prone TLS. Error-prone TLS is one of the fundamental mechanisms for genome mutagenesis. Benzo[a]pyrene (BP) is one of the common environmental pollutants generated during incomplete fuel combustion, in tobacco smoke, and in cooked food. BP can be metabolized in vivo to form the tumorigenic and mutagenic BPDE, which bind covalently with guanine residues in DNA. The formation of BPDE-dG lesion has been closely associated with lung cancer. To date, fifteen translesion synthesis polymerases have been identified in mammals, and DNA polymerase κ (Polκ) is the only known DNA polymerase in mouse or human cells that can bypass a BPDE-dG lesion efficiently and accurately, thereby reducing mutation risk. However, it is not known how a BPDE-dG lesion is accommodated and how dCTP is selected and incorporated by Polκ. In our work published in PNAS lately （2014,111(5), 1789-1794）, we have revealed for the first time that the role of Polκ's unique N-Clasp feature on the BPDE-dG bypass activity. However, the critical amino acid residues involved in the accommodation, incorporation, and extension past this lesion remain unknown. On the other hand, the human Aldo-Keto Reductases (AKRs) can also oxidaze BP to form the major metabolite BPQ. The metabolic activation pathways that lead to BPDE (the metabolites of P450) or BPQ (the metabolites of aldo-keto reductases) compete with each other. In contrast to the numerous investigations of the structural and biological characteristics of BPDE-DNA adducts, little attention, up till now, has been paid to the biophysical characterization of BPQ-DNA lesions and the carcinogenic and mutagenic contributions of these adducts in vivo. In addition, BPQ is a homobifunctional electrophile with aldehyde groups that can modify nucleophilic sites in DNA, proteins, and other cellular molecules. In this proposal, we will employ various techniques, including biochemistry, molecular biology, synthetic chemistry as well as x-ray crystallography to identify the critical amino acids in the accommodation, incorporation, and extension of BPDE-dG lesion, reveal the mutation spectrum of BPQ-DNA lesions and Polk's N-clasp feature in the formation of DNA-Polk crosslink (DPCs) derived from BPQ. Our results will deepen the understanding of the relationship between the polymerase kappa structural features and the TLS process, and target unique features of TLS polymerases in chemotherapy.

跨损伤DNA 合成(TLS)是细胞应答DNA 损伤的一种耐受机制。TLS 聚合酶可以在损伤的对面合成DNA。烟草、尾气排放所产生的苯并芘 (BP) 是一种常见的环境污染物。BP在体内经过代谢产生BPDE-dG的DNA损伤，与肺癌发生密切相关。目前在哺乳动物细胞中已发现大约15 种不同的TLS 聚合酶，Polk是迄今已知的唯一能正确跨越BPDE-dG损伤的聚合酶。我们在近期工作中首次发现Polk N-端独特的N-Clasp结构在调控跨越BPDE-dG加合物效应中的重要作用，但是关键位点氨基酸在跨损伤合成DNA中的调控机制还很不清楚。我们将应用生物化学、分子生物学、合成化学、X-射线晶体衍射等多种技术手段，阐明Polk正确跨越BPDE-dG反应的分子机理及Polk 参与形成BP衍生物介导的DNA-蛋白的交联。研究结果将加深理解Polk作为DNA损伤及响应修复的关键蛋白，其结构与功能之间的关系。

跨损伤DNA 合成(TLS)是细胞应答DNA 损伤的一种耐受机制，TLS 聚合酶可以在损伤的对面合成DNA。烟草、尾气排放所产生的苯并芘 (BP) 是一种常见的环境污染物。BP在体内经过代谢产生BPDE-dG的DNA损伤，与肺癌发生密切相关。目前在哺乳动物细胞中已发现大约15 种不同的TLS 聚合酶，Polk是迄今已知的唯一能正确跨越BPDE-dG损伤的聚合酶。我们首次发现Polk N-端独特的N-Clasp结构和N端21个氨基酸在调控跨越BPDE-dG加合物效应和基因组变异中发挥重要作用，影响聚合酶的保真性和延伸能力，为全面理解DNA聚合酶结构与功能之间的关系提供基础。而且我们明确了Polk在BPDE等暴露后可能参与多种DNA损伤应答途径，研究了Polk等TLS聚合酶招募到受阻复制叉处的调控机制，为研发以聚合酶为靶标的新的抗癌试剂、增强肿瘤细胞对化疗试剂的敏感性、降低肿瘤耐药性突变的产生提供重要的理论基础。受该基金的资助，目前已经发表SCI 文章7 篇，包括Nature Commun (2017)、Nucleic Acids Res (2018)等。