Oxidant and environmental toxicant-induced effects compromise ligation in DNA repair

氧化剂和环境毒物引起的影响会损害 DNA 修复中的连接

• 批准号: 9763551
• 负责人: MELIKE CAGLAYAN
• 金额: $ 24.35万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-15 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9763551
• 关键词: 8-hydroxyguanosine; APTX gene; Active Sites; Address; Affect; Applications Grants; Ataxia; Base Excision Repairs; Base Pairing; Biochemical; Biological Assay; Cell Death; Cell Line; Cell Survival; Cell-Mediated Cytolysis; Cells; Complement; Complex; Coupled; Coupling; Cultured Cells; DNA; DNA Damage; DNA Ligases; DNA Ligation; DNA Polymerase beta; DNA Repair; DNA Repair Pathway; DNA biosynthesis; DNA ligase I; DNA ligase III; DNA polymerase A; DNA-Directed DNA Polymerase; Data; Embryo; Enzymes; Exhibits; Failure; Fibroblasts; Genomic DNA; Genomic Instability; Goals; Guanine; Hand; Hypersensitivity; Impairment; Kinetics; Lead; Lesion; Ligase; Ligation; Link; Mammalian Cell; Measures; Mediating; Mentors; Molecular; Molecular Conformation; Mus; Mutation; Nucleotides; Oxidants; Oxidative Stress; Oxides; Pathway interactions; Phenotype; Polymerase; Process; Proteins; Reaction; Regulation; Research; Roentgen Rays; Role; Side; Site; Source; Structure; Testing; Toxic Environmental Substances; Toxicant exposure; Training; Work; adenylate; base; cell type; cytotoxic; cytotoxicity; endonuclease; environmental agent; experimental study; functional outcomes; in vivo; inhibitor/antagonist; mutant; overexpression; premature; prevent; public health relevance; recruit; repaired; stressor; success; tool; toxicant; tyrosyl-DNA phosphodiesterase

 DESCRIPTION (provided by candidate): Environmental and endogenous oxidants and toxicants can damage genomic DNA. Base excision repair (BER) is responsible for repairing such cytotoxic and mutagenic lesions that if not corrected can lead to deleterious mutations, genomic instability, or cell death. The multi-step BER pathway is coordinated by hand-off or channeling of DNA repair intermediates between the gap filling DNA synthesis step by DNA polymerase β (pol β) and ligation step by DNA ligase. However, pol β exhibits structural adjustments upon correct versus incorrect or oxidized nucleotide insertion into the gap and this impacts substrate channeling to the ligation step. The molecular mechanism of the hand-off to the ligation step of the BER pathway remains unclear. My preliminary data suggest that DNA ligase fails and abortive ligation occurs after pol β insertion of an incorrect or oxidized nucleotide. Furthermore, the modified structure of the resulting DNA intermediate after pol β mismatch extension coupled to gap filling leads to failed ligation. The goal of the proposed work is to examine whether ligation failure in the last step of BER as an important source of genomic instability and cytotoxicity in mammalian cells. In Aim 1, I will examine the effects of correct an incorrect nucleotide insertion during gap filling DNA synthesis coupled to ligation. For this purpose, I will measure nucleotide insertion kinetics in the presence of DNA ligase using wild-type pol β and active site mutants and then compare the rates and extents of ligation. I will use various types of DNA substrates to address the effects of insertion of oxidized or incorrect nucleotide. In Aim 2, I will evaluate the effects of other BER proteins, 3'-trimming enzymes, ligation conditions, and ligase forms for correcting or modifying failed ligation. I will evaluate other BER and 3'-end processing proteins in correcting impaired coordination during gap filling coupled ligation. Ligase reaction conditions and other forms of DNA ligase protein with impaired pol β interaction also will be examined for their roles in modifying failed ligation. In Aim 3, I ill determine the effects of DNA ligase deficiency on cellular cytotoxicity after oxidant and toxicant exposure. The possible link between cell phenotype and pol β-mediated oxidized base insertion with accumulation of toxic BER intermediates will be examined using in vivo cell survival assays. BER and ligation failure with environmental agent-induced cytotoxic lesions also will be quantified by measuring the amount of abortive ligation product in BER intermediates. Completion of these aims will increase the understanding of biochemical and cytotoxic effects of premature or failed DNA ligation during BER compromised by oxidant and environmental toxicant-induced effects.

 描述（由候选人提供）：环境和内源性氧化剂和毒物可损伤基因组DNA。碱基切除修复（BER）负责修复此类细胞毒性和致突变性病变，如果不进行纠正，可能会导致有害突变、基因组不稳定或细胞死亡。多步骤BER途径通过DNA修复中间体在DNA聚合酶β（pol β）的差距填充DNA合成步骤和DNA连接酶的连接步骤之间的传递或通道化来协调。然而，pol β在正确与不正确或氧化的核苷酸插入到差距中时表现出结构调整，这影响了底物引导到连接步骤。BER途径的连接步骤的传递的分子机制仍不清楚。我的初步数据表明，DNA连接酶失败，pol β插入不正确或氧化的核苷酸后，连接失败。此外，在pol β错配延伸后与缺口填充偶联的所得DNA中间体的修饰结构导致连接失败。这项工作的目的是研究BER最后一步的连接失败是否是哺乳动物细胞基因组不稳定性和细胞毒性的重要来源。在目标1中，我将检查在连接的间隙填充DNA合成期间正确的不正确的核苷酸插入的影响。为此，我将使用野生型pol β和活性位点突变体在DNA连接酶存在下测量核苷酸插入动力学，然后比较连接的速率和程度。我将使用各种类型的DNA底物来解决氧化或不正确的核苷酸插入的影响。在目标2中，我将评估其他BER蛋白、3 '-修剪酶、连接条件和连接酶形式对纠正或修饰失败的连接的影响。我将评估其他BER和3 '端加工蛋白在间隙填充偶联连接过程中纠正受损协调。还将检查连接酶反应条件和具有受损的pol β相互作用的其他形式的DNA连接酶蛋白在修饰失败的连接中的作用。目的3：研究DNA连接酶缺乏对氧化剂和毒物暴露后细胞毒性的影响。将使用体内细胞存活试验检查细胞表型和pol β介导的氧化碱基插入与毒性BER中间体蓄积之间的可能联系。BER和连接失败与环境因子诱导的细胞毒性损伤也将通过测量BER中间体中的失败连接产物的量来定量。这些目标的完成将增加理解生化和细胞毒性效应的过早或失败的DNA连接在BER损害氧化剂和环境毒物诱导的影响。