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

基本信息

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
项目类别：
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

项目摘要

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会损害基因组 DNA。碱基切除修复 (BER) 负责修复此类细胞毒性和诱变损伤，如果不纠正，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 聚合酶 β (pol β) 的间隙填充 DNA 合成步骤和 DNA 连接酶的连接步骤之间的 DNA 修复中间体的传递或通道来协调。 pol β 在正确与错误或氧化的核苷酸插入间隙时表现出结构调整，这会影响连接步骤的底物通道。我的初步数据表明，转移到连接步骤的分子机制仍不清楚。 DNA 连接酶失败，并且在 pol β 插入不正确或氧化的核苷酸后发生连接失败。此外，在 pol β 错配延伸与间隙填充耦合后所得 DNA 中间体的结构改变导致连接失败。拟议工作的目标是检查 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和细胞毒性的重要来源。在目标 1 中，我将检查在间隙期间纠正错误的核苷酸插入的影响。为此，我将使用野生型 pol β 和活性位点突变体测量 DNA 连接酶存在下的核苷酸插入动力学，然后比较连接的速率和程度。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' 末端加工蛋白在间隙填充偶联连接反应条件下纠正受损协调的情况，以及其他形式的 pol β 相互作用受损的 DNA 连接酶蛋白。还将检查它们在修饰失败连接中的作用，我将确定 DNA 连接酶缺陷对氧化剂和毒物暴露后细胞毒性的影响，以及细胞表型和 pol β 介导的氧化碱基插入与积累之间的可能联系。将使用体内细胞存活测定来检查有毒 BER 中间体的数量，并且还将通过测量中失败的连接产物的量来量化环境因素诱导的细胞毒性损伤的连接失败。 BER 中间体的完成将增加对 BER 期间因氧化剂和环境毒物引起的影响而导致的过早或失败的 DNA 连接的生化和细胞毒性影响的理解。