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: 10582554
• 负责人: Bennett Van Houten
• 金额: $ 90.63万
• 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/10582554
• 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.

标题：观察碱和核苷酸切除修复蛋白之间的合作相互作用。 PI：Bennett Van Houten，博士 摘要/摘要 这个高度创新的项目旨在回答有关DNA的几个基本问​​题 修复蛋白质可保护人类基因组免受环境诱导的DNA损伤。这 项目使用合并生物化学，单分子分析和高度的集成方法 具有高时空和空间分辨率研究的创新化学遗传学细胞生物学工具 DNA修复过程中的分子交接。我们认为关键核苷酸切除修复蛋白包括 UV-DDB，XPA和XPC-RAD23B以动态方式使用特定的碱基切除修复蛋白到 在染色质的背景下，过程氧化碱。特别是，我们将遵循纯化的DNA修复 蛋白质和/或蛋白质从核提取物上标记的蛋白质在裸露位点相互作用 DNA和染色体DNA使用DNA钢干光学平台。以初步数据为基础 UV-DDB可以在核小体的背景下更改特定病变的寄存器的前提，我们 将测试范式转移假设，即UV-DDB与其他NER蛋白一起工作是 一般损伤传感器，可以刺激APE1和11个哺乳动物DNA糖基酶在 它们各自的氧化DNA底物。该项目将开发并验证新的基因组工具 在整个基因组的定义位点上放置8-oxog加合物，以评估染色质结构 和染色质重塑作用修复。我们还将开发和使用几个高分辨率 荧光方法，包括单个粒子跟踪方案（基于晕圈和快照） 观察单个修复蛋白到达，并在活细胞中实时处理损伤位点。最后，我们 认为UV-DDB和XPC-RAD23B与胸腺氨酸DNA糖基酶一起使用以改变甲基化模式 在细胞中，最终改变基因表达谱。这些方法一起将给予 修复过程中DNA损伤处理的复杂过程的前所未有的观点 关于损害识别的几个关键问题，在没有 超分辨率方法。该项目的完成将对该领域产生较长而持久的影响。