Investigation of DNA base excision repair

DNA碱基切除修复的研究

• 批准号: 9916777
• 负责人: Zucai Suo
• 金额: $ 30.8万
• 依托单位: FLORIDA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-07 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9916777
• 关键词: 5' -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-(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

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.

项目总结 基因组DNA不断地受到内源性或外源性因素或过程的破坏。单元格 已经进化出几种DNA修复途径，包括DNA碱基切除修复(BER)来修复 损坏。BER修复绝大多数单碱基损伤，包括主要的氧化DNA 病变8-氧鸟嘌呤(8-oxoG)。8-oxoG具有高度的突变性，因为它能够形成一个Watson- 具有正确dCTP的Crick碱基对和具有错误dATP的Hoogsteen碱基对 跨病变DNA合成(TLS)，这可能导致癌症。为了修复8-oxoG，人类细胞使用 四种酶进行连续的BER反应，它们是人8-oxoG DNA糖基酶 (HOGG_1)、AP内切酶(APE_1)、DNA聚合酶β(HPOL_β)和DNA连接酶III/XRCC1。 尽管这些酶已经被研究了多年，但仍有许多未解决的或 关于它们的催化作用的有争议的机械问题。例如，它不是 完全清楚的是，具有DNA聚合酶活性和5‘-脱氧核糖的Hpol1β- 5-磷酸裂解酶(5-ʹ裂解酶)活性，既执行缺口DNA的合成和5-磷酸裂解酶的去除 误码率期间的DRP。此外，新发现的第三二价金属离子在HPOLβ中的作用-- 催化的核苷酸掺入仍不清楚。此外，hOGG1- 催化的碱基切除和链断裂仍然存在争议。最后，提出了一种新的可持续发展战略 在BER蛋白之间必须有某种协调机制，以允许高效和 DNA损伤的快速修复。首席调查员(PI)的长期目标是 使用生化和生物物理方法来阐明详细的动力学和结构 BER酶及蛋白质/蛋白质和蛋白质/DNA动态相互作用的机制 这条重要的DNA修复途径。在本提案的目标1中，国际和平研究所将采用常规和 依赖时间的结晶学为HPOLβ的dRPase活性建立结构基础， 明确第三种二价金属离子的作用，观察TLS的病变旁路和延伸步骤 用HPOLβ对8-oxoG进行催化，并对其催化机理进行了细化。在目标2中，PI将使用 单分子Förster共振能量转移(FRET)和共局域单分子 用光谱学(COSMOS)研究hOGG1与APE1和TO的相互作用 阐明了误码率协调的机制。完成这项提案将更好地定义 HPOLβdRPase结构域和hOGG1中关键活性中心残基的催化作用 第三价金属离子在DNA聚合酶催化中作用的新信息 反应，并为误码率期间的协调模式提供了前所未有的证据。