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）：环境和内源性氧化物和毒物会损害基因组DNA。基本惊喜修复（BER）负责修复这种细胞毒性和诱变病变，如果无法校正，可能会导致有害突变，基因组不稳定性或细胞死亡。多步BER途径通过DNA填充DNA合成之间的DNA修复中间体的交接或通道通过DNA聚合酶β（POLβ）和DNA连接酶的结扎步骤来协调。但是，POLβ在正确的与错误或氧化的核OTIDE插入间隙时表现出结构调整，这会影响底物向连接步骤的传递。交接到BER途径的结扎步骤的分子机制尚不清楚。我的初步数据表明，DNA连接酶失败，并且在polβ插入不正确或氧化的核丁基后发生流产。此外，POLβ不匹配延伸后所得的DNA中间体的修饰结构与间隙填充耦合导致连接失败。拟议工作的目的是检查BER的最后一步的结扎失败是否是哺乳动物细胞中基因组不稳定性和细胞毒性的重要来源。在AIM 1中，我将检查在填充DNA合成的间隙填充与结扎的差异过程中正确核定插入的效果。为此，我将使用野生型POLβ和活性位点突变体在存在DNA连接酶的情况下测量核苷酸插入动力学，然后比较连接的速率和范围。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AIM 2中，我将评估其他BER蛋白，3'-Trimming酶，连接条件和连接酶形式的影响，以纠正或修改失败的连接。我将评估其他BER和3'末端加工蛋白，以纠正间隙填充耦合连接期间的协调受损。连接酶反应条件和其他形式的DNA连接酶蛋白与受损的POLβ相互作用将检查其在修饰失败连接中的作用。在AIM 3中，我确定了DNA连接酶缺乏对氧化剂和毒性暴露后细胞细胞毒性的影响。细胞表型与POLβ介导的氧化碱插入与有毒BER中间体的准确性之间的可能联系将使用体内细胞存活分析进行检查。通过测量BER中间体中流产的连接产物的量，还将量化与环境剂诱导的细胞毒性病变有关的BER和连接破坏。这些目标的完成将增加对因氧化剂和环境有毒物质诱导的作用而损害的过早DNA连接的生化和细胞毒性作用的理解。