Watching cooperative interactions between base and nucleotide excision repair proteins

标题：观察碱基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试图回答几个基本的问题，即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 该项目使用了一种结合生物化学、单分子分析和高度 创新的化学发生细胞生物学工具，以高时间和空间分辨率进行研究 DNA修复过程中的分子交接。我们假设关键的核苷酸切除修复蛋白包括 UV-DDB、XPA和XPC-RAD23B与特定的碱基切除修复蛋白动态作用 在染色质的背景下处理氧化碱。特别是，我们将跟踪纯化的DNA修复 蛋白质和/或从核抽提物标记的蛋白质，因为它们在裸露的损伤部位相互作用 DNA和使用DNA钢索光学平台的显色化DNA。以初步数据为基础， 假设UV-DDB可以改变核小体背景下的特定病变的登记，我们 将测试范式转换假设，即UV-DDB与其他NER蛋白协同工作是一种 一般损伤感受器，并可刺激APE1和11哺乳动物DNA糖基酶活性 它们各自氧化的DNA底物。该项目将开发和验证新的基因组工具以 将8-oxoG加合物放置在整个基因组中的特定位置以评估染色质结构 染色质重构体可以起到修复作用。我们还将开发和使用几个高分辨率 荧光方法包括单粒子跟踪协议(基于Halo和SNAP标记)以 观察单个修复蛋白到达并实时处理活细胞中的损伤位置。最后，我们 假设UV-DDB和XPC-RAD23B与胸腺嘧啶DNA糖基酶共同作用改变甲基化模式 并最终改变基因表达谱。这些方法加在一起将提供一个 修复和回答过程中DNA损伤处理的复杂过程的前所未有的视角 关于损害识别的几个关键问题，在没有 超分辨率即将到来。该项目的完成将对实地产生长期和持久的影响。

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.