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: 8582552
• 负责人: ANEEL K. AGGARWAL
• 金额: $ 43.93万
• 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 2017-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8582552
• 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; 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）：Translesion合成（TLS）DNA聚合酶（POLS）通过DNA病变促进复制，从而阻止复制叉的持续进展。尽管使用各种酵母和人类TLS pol的结构研究表明它们可以以高度专业化的方式在TLS中起作用，但可用于的信息可用于 它们的生物学作用相对微薄。特别是POL的生物学作用？仍然是最不了解的。在拟议的研究中，我们将使用合并的遗传，生化和结构方法来检验POL的假设？通过损害Watson-Crick（W-C）碱基配对或向DNA小凹槽突出的DNA病变做出重要贡献。此外，最重要的是，将进行研究以检验POL的假设？与未损坏的残基相比，与DNA病变相对的功能与DNA病变相反。在AIM 1中，我们将研究POL的作用？以及通过N1-甲基腺嘌呤（1-MEA）促进复制的人类细胞中的其他TLS POL，这会损害W-C碱基配对； N3-甲基腺嘌呤（3-DMEA）的Deaza衍生物，该腺苷（3-DMEA）突出到DNA小凹槽中；和1,3-丁二烯（N2-DG，R-丁二烯单氧化物）的N2-DG加合物，类似于3-DMEA，是一个较小的凹槽病变，但化学上更为复杂。 1-MEA和3-DMEA是通过暴露于环境烷基化剂和内源性细胞反应而产生的，而1,3-丁二烯是一种重要的工业化学化学物质和环境污染物。对于人类细胞中的TLS分析，我们将利用两个不同的双链质粒系统，一个基于SV40的基于SV40的质粒和基于EBV的质粒，以及POL的相对贡献？并将确定病变旁路和诱变性的其他pol。在AIM 2中，将进行生化研究以检查POL的水平吗？与1-MEA，3-DMEA和N2-DG R丁二烯单氧化物加合物相对的合成DNA中的其他POL。通过稳态动力学分析，我们将确定POL的催化效率和保真度？以及其他用于插入核苷酸（NT）与每个病变相对的核苷酸（NT），并从插入的NT延伸的POL。在AIM 3中，POL的结构？在带有1-MEA，3-DMEA，N2-DG R丁二烯单氧化物的三元络合物中，还将确定（6-4）TT光productuct以发现POL的底部？能够以主要无误差方式与这些DNA病变相反。将进行生化研究和人类细胞中的TLS研究，以检查残基中突变的作用，这些突变有助于稳定病变部位对面的正确传入的NT。拟议的研究与癌症生物学和癌症病因高度相关，因为它们将揭示Pol？与其他TLS POL的结合，促进了与多种DNA加合物相对的TLS的主要无错误模式。 TLS的无错误模式与POL的角色保持一致吗？在抑制癌变中，否则会导致暴露于环境和化学致癌物。