Role of human DNA polymerase iota in replicative bypass of DNA lesions

人类DNA聚合酶iota在DNA损伤复制旁路中的作用

• 批准号: 8762244
• 负责人: ANEEL K. AGGARWAL
• 金额: $ 44.37万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-11-15 至 2015-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8762244
• 关键词: 1,3-Butadiene; 3,4-epoxy-1-butene; Active Sites; Adenine; Adoption; Alkylating Agents; Base Pairing; Biochemical; Biological; Butadiene; Bypass; Cancer Biology; Cancer Etiology; Cells; Chemicals; Complex; DNA; DNA Adducts; DNA Damage; DNA Structure; DNA lesion; DNA polymerase iota; DNA-Directed DNA Polymerase; Ensure; Environmental Carcinogens; Environmental Pollutants; Environmental Pollution; Exposure to; Family; Food; Genetic; Genetic study; Geometry; Human; Human Herpesvirus 4; Incidence; Kinetics; Lesion; Minor Groove; Molecular Conformation; Mutagenesis; Mutation; Nucleotides; Occupational; Plasmids; Polymerase; Purine Nucleotides; Purines; Reaction; Relative (related person); Replication Origin; Role; Shapes; Simian virus 40; Site; Structure; System; Testing; UV induced; Virus Replication; Yeasts; adduct; base; carcinogenesis; chemical carcinogen; environmental chemical; human DNA; in vivo; public health relevance; purine

DESCRIPTION (provided by applicant): Translesion synthesis (TLS) DNA polymerases (Pols) promote replication through DNA lesions which block the continued progression of the replication fork. Although structural studies with the various yeast and human TLS Pols have indicated that they could function in TLS in highly specialized ways, the information available for their biological roles has been relatively meager; in particular, the biological role of Pol? has remained the least understood. In the proposed studies, we will use a combined genetic, biochemical, and structural approach to test the hypothesis that Pol? makes an important contribution to promoting replication through DNA lesions which impair Watson-Crick (W-C) base pairing or which protrude into the DNA minor groove. Furthermore, and most importantly, studies will be done to test the hypothesis that Pol? functions opposite DNA lesions in a much more error-free manner than opposite undamaged residues. In Aim 1, we will examine the roles of Pol? and other TLS Pols in human cells in promoting replication through N1-methyl adenine (1-MeA), which impairs W-C base pairing; a deaza derivative of N3-methyl adenine (3-dMeA), which protrudes into the DNA minor groove; and an N2-dG adduct of 1,3-butadiene (N2-dG, R-butadiene monoepoxide), which like 3-dMeA, is a minor groove lesion but chemically more complex. 1-MeA and 3-dMeA are generated from exposure to environmental alkylating agents and from endogenous cellular reactions, and 1,3-butadiene is an important industrial chemical and an environmental pollutant. For TLS analysis in human cells, we will utilize two different duplex plasmid systems, an SV40 origin-based plasmid and an EBV origin-based plasmid, and the relative contributions of Pol? and of other Pols to lesion bypass and to mutagenicity will be determined. In Aim 2, biochemical studies will be done to examine the proficiency of Pol? and of other Pols in synthesizing DNA opposite the 1-MeA, 3-dMeA, and N2-dG R-butadiene monoepoxide adducts. By steady-state kinetic analyses, we will determine the catalytic efficiency and fidelity of Pol? and of other Pols for inserting a nucleotide (nt) opposite each of these lesions and for extending from the inserted nt. In Aim 3, structures of Pol? in ternary complex with the 1-MeA, 3-dMeA, N2-dG R-butadiene monoepoxide, and also a (6-4) TT photoproduct will be determined to uncover the bases of Pol? ability to function opposite these DNA lesions in a predominantly error-free manner. Biochemical studies, and TLS studies in human cells, will be carried out to examine the effects of mutations in residues that help stabilize the correct incoming nt opposite the lesion site. The proposed studies are highly relevant for cancer biology and cancer etiology as they will reveal whether Pol?, in conjunction with other TLS Pols, promotes a predominantly error-free mode of TLS opposite a diverse array of DNA adducts. An error-free mode of TLS would be in keeping with a role for Pol? in suppression of carcinogenesis that would otherwise result from exposure to environmental and chemical carcinogens.

描述（由申请人提供）：跨损伤合成（TLS）DNA聚合酶（Pols）通过DNA损伤促进复制，DNA损伤阻止复制叉的持续进展。尽管对各种酵母和人类 TLS Pol 的结构研究表明它们可以以高度专业化的方式在 TLS 中发挥作用，但现有的信息 它们的生物学作用相对微弱；特别是 Pol 的生物学作用？仍然是最不被理解的。在拟议的研究中，我们将使用遗传、生化和结构相结合的方法来检验 Pol？通过损害 Watson-Crick (W-C) 碱基配对或突出到 DNA 小沟的 DNA 损伤，对促进复制做出了重要贡献。此外，最重要的是，将进行研究来检验 Pol？与未受损残基相比，它以更无差错的方式对抗 DNA 损伤。在目标 1 中，我们将研究 Pol? 的角色？和人类细胞中的其他 TLS Pols 通过 N1-甲基腺嘌呤 (1-MeA) 促进复制，这会损害 W-C 碱基配对； N3-甲基腺嘌呤 (3-dMeA) 的脱氮衍生物，突出到 DNA 小沟中； 1,3-丁二烯的 N2-dG 加合物（N2-dG，R-丁二烯单环氧化物）与 3-dMeA 一样，是一种小沟病变，但化学性质更复杂。 1-MeA和3-dMeA是通过暴露于环境烷化剂和内源性细胞反应而产生的，1,3-丁二烯是一种重要的工业化学品和环境污染物。对于人类细胞中的 TLS 分析，我们将利用两种不同的双链体质粒系统，即基于 SV40 起源的质粒和基于 EBV 起源的质粒，以及 Pol？以及其他Pols对病变绕过和致突变性的影响将被确定。在目标 2 中，将进行生化研究来检查 Pol? 的熟练程度。以及与 1-MeA、3-dMeA 和 N2-dG R-丁二烯单环氧化物加合物相反的 DNA 合成中的其他 Pols。通过稳态动力学分析，我们将确定 Pol? 的催化效率和保真度。以及用于在这些损伤中的每一个相对处插入核苷酸(nt)并用于从插入的nt延伸的其他Pols。在目标 3 中，Pol 的结构？与 1-MeA、3-dMeA、N2-dG R-丁二烯单环氧化物以及 (6-4) TT 光产物的三元复合物将被确定以揭示 Pol? 的碱基。能够以基本无差错的方式对抗这些 DNA 损伤。将在人类细胞中进行生化研究和 TLS 研究，以检查残基突变的影响，这些残基有助于稳定病变部位对面的正确传入 nt。拟议的研究与癌症生物学和癌症病因学高度相关，因为它们将揭示 Pol? 是否与其他 TLS Pol 结合，促进与多种 DNA 加合物相反的 TLS 的主要无错误模式。 TLS 的无错误模式是否符合 Pol 的角色？抑制因暴露于环境和化学致癌物而导致的致癌作用。