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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：
9982953
负责人：
MELIKE CAGLAYAN
金额：
$ 23.78万
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 2022-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9982953
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-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破坏基因组DNA。碱基切除修复(BER)负责修复这些细胞毒性和突变损伤，如果不纠正这些损伤可能会导致有害突变、基因组不稳定或细胞死亡。多步误码率途径是通过DNA聚合酶β(PolIβ)填补缺口的合成步骤和DNA连接酶连接步骤之间的DNA修复中间产物的传递或通道来协调的。然而，POLβ在正确与不正确或被氧化的核苷酸插入缺口时显示出结构调整，这会影响底物向连接步骤的通道。BER通路的连接步骤的分子机制尚不清楚。我的初步数据表明，在POLβ插入错误或氧化的核苷酸后，dna连接酶失效，并发生流产连接。此外，POLβ错配延伸后的中间产物与缺口填充耦合后的结构改变导致连接失败。这项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，我将检查在连接连接的间隙填充DNA合成过程中纠正错误核苷酸插入的效果。为此，我将使用野生型POLβ和活性位点突变体来测量DNA连接酶存在下的核苷酸插入动力学，然后比较连接的速度和程度。我将使用各种类型的DNA底物来解决插入氧化或错误核苷酸的影响。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改失败的连接的影响。我将评估其他BER和3‘端加工蛋白在纠正缝隙填充偶联结扎过程中受损的协调性方面的作用。连接酶反应条件和其他形式的具有受损的POLβ相互作用的DNA连接酶蛋白也将被检查它们在修改失败的连接过程中的作用。在目标3中，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。细胞表型和POLβ介导的氧化碱基插入与有毒的误码率中间产物的积累之间的可能联系将通过体内细胞存活分析来检验。BER和结扎失败与环境因素引起的细胞毒性损害也将通过测量BER中间产物中流产的结扎产物的数量来量化。这些目标的完成将增加对过早或失败的DNA连接在BER过程中因氧化剂和环境毒物诱导的影响而产生的生化和细胞毒性影响的理解。