Watching cooperative interactions between base and nucleotide excision repair proteins

观察碱基和核苷酸切除修复蛋白之间的协同相互作用

• 批准号: 10763255
• 负责人: Bennett Van Houten
• 金额: $ 1.31万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2028-02-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10763255
• 关键词: 8-hydroxyguanosine; Base Excision Repairs; Biochemistry; Cells; Cellular biology; Chromatin; Chromatin Structure; Complex; DNA; DNA Damage; DNA Repair; DNA Repair Enzymes; DNA Repair Gene; DNA glycosylase; DNA purification; Data; Doctor of Philosophy; Environment; Functional disorder; Gene Expression; Genome; Human Genome; Individual; Inflammation; Label; Lesion; Malignant Neoplasms; Molecular; Nerve Degeneration; Nuclear Extract; Nucleosomes; Nucleotide Excision Repair; Nucleotides; Optics; Pathway interactions; Premature aging syndrome; Process; Proteins; Protocols documentation; RAD23B gene; Resolution; Site; Testing; Thymine DNA Glycosylase; Time; Work; XPA gene; adduct; base; environmental mutagens; genomic locus; genomic tools; innovation; methylation pattern; oxidative DNA damage; particle; repaired; response; sensor; single molecule; tool; ultra high resolution

Title: Watching cooperative interactions between base and nucleotide excision repair proteins. PI: Bennett Van Houten, PhD Abstract/Summary This highly innovative project seeks to answer several fundamental questions regarding how DNA repair proteins work to protect the human genome from environmentally-induced DNA damage. This project uses an integrated approach combining biochemistry, single molecule analysis and highly innovative chemoptogenetic cell biology tools to study with high temporal and spatial resolution molecular hand-offs during DNA repair. We posit that key nucleotide excision repair proteins including UV-DDB, XPA, and XPC-RAD23B work in a dynamic way with specific base excision repair proteins to process oxidized bases in the context of chromatin. Specially, we will follow purified DNA repair proteins and/or proteins labeled from nuclear extracts as they interact at sites of damage on naked DNA and chromatinized DNA using a DNA tightrope optical platform. Building on preliminary data and premise that UV-DDB can change the register of specific lesions in the context of the nucleosome, we will test the paradigm shifting hypothesis that UV-DDB working in concert with other NER proteins is a general damage sensor and can stimulate APE1 and 11 mammalian DNA glycosylases activities on their respective oxidized DNA substrates. This project will develop and validate new genomic tools to place 8-oxoG adducts at defined sites throughout the genome to assess the how chromatin structure and chromatin remodelers effects repair. We will also develop and use several high-resolution fluorescent approaches including single particle tracking protocols (based on Halo- and SNAP-tags) to watch individual repair proteins arrive and process damage sites in real-time in living cells. Finally, we posit that UV-DDB and XPC-RAD23B work with thymine DNA glycosylase to alter methylation patterns in cells and ultimately change gene expression profiles. Together these approaches will give an unprecedented view of the complex process of DNA damage processing during repair and answer several key questions regarding damage recognition that have been intractable in the absence of super-resolution approaches. Completion of this project will have a long and lasting impact on the field.

观察碱基和核苷酸切除修复蛋白之间的合作相互作用。 PI：班尼特货车Houten，博士 摘要/概要 这个高度创新的项目旨在回答关于DNA如何 修复蛋白的作用是保护人类基因组免受环境诱导的DNA损伤。这 该项目采用综合的方法，结合生物化学，单分子分析和高度 创新的化学光遗传学细胞生物学工具，以高时间和空间分辨率进行研究 DNA修复过程中的分子传递我们发现，关键的核苷酸切除修复蛋白， UV-DDB、XPA和XPC-RAD 23 B以动态方式与特异性碱基切除修复蛋白一起工作， 处理染色质中的氧化碱基。特别地，我们将跟踪纯化的DNA修复 蛋白质和/或标记自核提取物的蛋白质，因为它们在裸细胞损伤部位相互作用， DNA和染色质化的DNA，使用DNA tightrope光学平台。在初步数据的基础上， 假设UV-DDB可以改变核小体中特定病变的登记，我们 将测试范式转移假说，即UV-DDB与其他NER蛋白协同工作是一种新的蛋白质。 一般的损伤传感器，可以刺激APE 1和11哺乳动物DNA糖基化酶的活性， 它们各自的氧化DNA底物。该项目将开发和验证新的基因组工具， 将8-oxoG加合物放置在整个基因组的指定位点，以评估染色质结构如何 和染色质重塑影响修复。我们还将开发和使用几种高分辨率的 荧光方法，包括单粒子跟踪协议（基于Halo和SNAP标签）， 观察单个修复蛋白到达并实时处理活细胞中的损伤部位。最后我们 证明UV-DDB和XPC-RAD 23 B与胸腺嘧啶DNA糖基化酶一起改变甲基化模式 并最终改变基因表达谱。这些方法将共同提供一个 对DNA损伤修复和应答过程中复杂的处理过程的前所未有的看法 关于损害承认的几个关键问题， 超分辨率方法。该项目的完成将对实地产生长期和持久的影响。