Investigation of DNA base excision repair

DNA碱基切除修复的研究

基本信息

批准号：
9757781
负责人：
Zucai Suo
金额：
$ 30.75万
依托单位：
FLORIDA STATE UNIVERSITY
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：
2018-08-07 至 2022-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9757781
关键词：
5&apos -deoxyribose phosphate lyase 8-Oxoguanine DNA Glycosylase 8-hydroxyguanosine Acids Active Sites Adenine Base Excision Repairs Base Pairing Binding Biochemical Buffers Bypass Catalysis Cell Extracts Cells Complex Crystallography DNA DNA Damage DNA Polymerase beta DNA Repair DNA Repair Pathway DNA biosynthesis DNA glycosylase DNA lesion DNA ligase I DNA ligase III DNA polymerase A DNA-(apurinic or apyrimidinic site) lyase DNA-Directed DNA Polymerase DNA-Protein Interaction Deoxyribose Enzymes Excision Fluorescence Resonance Energy Transfer Genomic DNA Human In Vitro Individual Investigation Ions Kinetics Label Lead Lesion Licensing Malignant Neoplasms Metal Ion Binding Metals Methodology Methods Modeling Molecular Molecular Conformation N-terminal Nucleotides Pathway interactions Polymerase Principal Investigator Process Proteins Protons Reaction Repair Complex Research Personnel Role Schiff Bases Site Spectrum Analysis Spliceosome Assembly Pathway Structure Sulfur System Testing Time Transcription Initiation Vertebral column X-Ray Crystallography XRCC1 gene base biophysical techniques crosslink divalent metal enzyme mechanism fluorophore human DNA inorganic phosphate member nucleotide analog protein complex reconstitution repair enzyme repaired seal single molecule small molecule time use

项目摘要

PROJECT SUMMARY Genomic DNA is constantly damaged by endogenous or exogenous agents or processes. Cells have evolved several DNA repair pathways including DNA base excision repair (BER) to fix the damage. BER repairs the vast majority of single base lesions, including a major oxidative DNA lesion 8-oxoguanine (8-oxoG). 8-oxoG is highly mutagenic due to its ability to form both a Watson- Crick base pair with correct dCTP and a Hoogsteen base pair with incorrect dATP during translesion DNA synthesis (TLS), which can lead to cancer. To repair 8-oxoG, human cells use four enzymes to carry out sequential BER reactions and they are human 8-oxoG DNA glycosylase (hOGG1), AP endonuclease (APE1), DNA polymerase β (hPolβ), and DNA ligase III/XRCC1. Although these enzymes have been investigated for years, there are many unresolved or controversial mechanistic questions about their catalytic functions. For example, it is not completely clear how hPolβ, which possesses a DNA polymerase activity and a 5′-deoxyribose- 5-phosphate lyase (dRPase) activity, executes both gap-filling DNA synthesis and removal of 5ʹ- dRP during BER. Moreover, the role of a newly discovered third divalent metal ion in hPolβ- catalyzed nucleotide incorporation remains unclear. Additionally, the mechanisms of hOGG1- catalyzed base excision and strand scission remain controversial. Finally, it has been proposed that there must be some mechanism of coordination between BER proteins to allow efficient and rapid repair of DNA damages. The long-term objectives of the Principle Investigator (PI) are to use biochemical and biophysical methods to elucidate the detailed kinetic and structural mechanisms for the BER enzymes and dynamic protein/protein and protein/DNA interactions in this important DNA repair pathway. In Aim 1 of this proposal, the PI will employ conventional and time-dependent crystallography to establish the structural basis for the dRPase activity of hPolβ, define the role of the third divalent metal ion, watch the lesion bypass and extension steps of TLS across 8-oxoG by hPolβ, and refine the catalytic mechanism of hOGG1. In Aim 2, the PI will use single-molecule Förster resonance energy transfer (FRET) and colocalization single-molecule spectroscopy (CoSMoS) to characterize the interaction between hOGG1 and APE1 and to elucidate the mechanism of BER coordination. Completion of this proposal will better define the catalytic roles of critical active site residues in the hPolβ dRPase domain and hOGG1, provide new information for the role of the third divalent metal ion in the DNA polymerase-catalyzed reaction, and give unprecedented evidence for the modes of coordination during BER.

项目概要基因组 DNA 不断受到内源性或外源性物质或细胞过程的损伤。已经进化出多种 DNA 修复途径，包括 DNA 碱基切除修复 (BER)，以修复 BER 修复绝大多数单碱基损伤，包括主要的氧化 DNA 损伤 8-氧代鸟嘌呤 (8-oxoG) 8-oxoG 具有高度致突变性，因为它能够形成 Watson-。具有正确 dCTP 的克里克碱基对和具有错误 dATP 的 Hoogsteen 碱基对人类细胞使用可导致癌症的跨损伤 DNA 合成 (TLS) 来修复 8-oxoG。进行连续 BER 反应的四种酶，它们是人 8-oxoG DNA 糖基化酶 (hOGG1)、AP 核酸内切酶 (APE1)、DNA 聚合酶 β (hPolβ) 和 DNA 连接酶 III/XRCC1。尽管这些酶已被研究多年，但仍有许多未解决或未解决的问题例如，关于其催化功能的有争议的机械问题不是这样。完全清楚 hPolβ 是如何具有 DNA 聚合酶活性和 5'-脱氧核糖- 5-磷酸裂解酶 (dRPase) 活性，执行间隙填充 DNA 合成和 5ʹ- 去除此外，新发现的第三种二价金属离子在 hPolβ- 中的作用。此外，hOGG1- 催化核苷酸掺入的机制仍不清楚。最后，催化碱基切除和链断裂仍然存在争议。 BER 蛋白之间必须存在某种协调机制，以实现高效且快速修复 DNA 损伤。首席研究员 (PI) 的长期目标是使用生物化学和生物物理方法来阐明详细的动力学和结构 BER 酶和动态蛋白质/蛋白质和蛋白质/DNA 相互作用的机制在该提案的目标 1 中，PI 将采用常规和修复途径。时间依赖性晶体学建立 hPolβ dRPase 活性的结构基础，定义第三个二价金属离子的作用，观察TLS的病变旁路和延伸步骤通过 hPolβ 跨越 8-oxoG，并完善 hOGG1 的催化机制在目标 2 中，PI 将使用。单分子 Förster 共振能量转移 (FRET) 和共定位单分子光谱 (CoSMoS) 来表征 hOGG1 和 APE1 之间的相互作用并阐明 BER 协调机制。完成本提案将更好地定义 BER 协调机制。 hPolβ dRPase 结构域和 hOGG1 中关键活性位点残基的催化作用，提供关于第三种二价金属离子在 DNA 聚合酶催化中的作用的新信息反应，并为 BER 期间的协调模式提供前所未有的证据。