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: 9182819
• 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 2018-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9182819
• 关键词: 1,3-Butadiene; 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 biosynthesis; DNA lesion; DNA polymerase iota; DNA replication fork; DNA-Directed DNA Polymerase; Ensure; Environmental Carcinogens; Environmental Pollutants; Environmental Pollution; Exposure to; Family; Food; Genetic; Genetic study; Geometry; Human; Human Herpesvirus 4; Impairment; Incidence; Income; Industrialization; Kinetics; Lesion; Minor Groove; Molecular Conformation; Mutagenesis; Mutation; Nucleotides; Occupational; Plasmids; Purine Nucleotides; Purines; Reaction; Replication Origin; Role; Shapes; Simian virus 40; Site; Structure; System; Testing; UV induced; Yeasts; adduct; base; carcinogenesis; chemical carcinogen; environmental chemical; human DNA; in vivo; public health relevance

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）：转录合成（TLS）DNA聚合酶（Pos）通过DNA损伤促进复制，DNA损伤阻断复制叉的继续进展。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，但可用于研究的信息仍然不足。 他们的生物作用一直相对微薄，特别是，生物作用的波尔？仍然是人们最不了解的。在拟议的研究中，我们将使用一个组合的遗传，生物化学和结构的方法来测试的假设，波尔？通过损害Watson-Crick（W-C）碱基配对或突出到DNA小沟中的DNA损伤对促进复制做出重要贡献。此外，最重要的是，研究将进行测试的假设，波尔？相对于相对的未受损残基，相对的DNA损伤以更无差错的方式起作用。在目标1中，我们将研究波尔的作用？和人类细胞中的其他TLS Pol通过N1-甲基腺嘌呤（1-MeA）促进复制，其损害W-C碱基配对; N3-甲基腺嘌呤的脱氮衍生物（3-dMeA），其突出到DNA小沟中;和1，3-丁二烯的N2-dG加合物（N2-dG，R-丁二烯单环氧化物），其像3-dMeA一样，是小沟损伤，但化学上更复杂。1-MeA和3-dMeA是由暴露于环境烷基化剂和内源性细胞反应产生的，并且1，3-丁二烯是重要的工业化学品和环境污染物。对于TLS分析在人类细胞中，我们将利用两种不同的双链体质粒系统，一个SV 40起源为基础的质粒和EBV起源为基础的质粒，和相对贡献的Pol？以及其他Pol对病变旁路和致突变性的影响。在目标2中，将进行生化研究，以检查Pol？和其它Pol在合成DNA中与1-MeA、3-dMeA和N2-dGR-丁二烯单环氧化物加合物相反。通过稳态动力学分析，我们将确定的催化效率和保真度的波尔？以及其它Pol，用于插入与这些损伤中的每一个相对的核苷酸（nt）并从插入的nt延伸。在目标3，结构的Pol？与1-MeA、3-dMeA、N2-dG R-丁二烯单环氧化物以及（6-4）TT光产物的三元络合物将被确定以揭示Pol？以主要无差错的方式对抗这些DNA损伤的能力。将进行生物化学研究和人类细胞中的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？，与其他TLS Pol结合，促进了TLS的主要无错误模式，与各种各样的DNA加合物相反。TLS的无错误模式将与Pol？抑制致癌作用，否则将导致暴露于环境和化学致癌物。