Structure and Function of DNA Repair Enzymes

描述（由申请人提供）：我们的假设是，我们研究的酶家族的缺陷导致了异常的碱基切除和同源定向修复，这是驱动人类致癌的引擎。大多数内源性和辐射诱导的DNA损伤被BER去除，当这一途径失败时，产生的底物被引导到HDR中。本项目的总体目标是在原子水平上了解DNA修复酶的三个家族，DNA糖基酶的HhH和Fpg/Nei家族以及重组酶的RecA-RAD51家族如何识别和处理它们的底物，以及这些蛋白质的种系和肿瘤相关变体如何分别影响癌症易感性和致癌作用。基于我们在生物化学和结构生物学方面的优势，我们的项目现在是由人类碱基切除修复和HDR酶的生殖系和肿瘤相关变体的鉴定和表征所驱动的。核心A将根据结构和系统发育鉴定氧化DNA糖基酶和RAD51的人类种系和体细胞DNA序列变体。项目1将证明这些修复变异体是否会诱导细胞转化，在小鼠细胞中是否具有诱变性，以及它们是否会影响细胞对电离辐射和化疗药物的反应。项目2将检查氧化糖基化酶变体的生化特性，并利用底物和适当的糖基化酶变体解决野生型酶的结构。项目3将检查RAD51变体的生化和适当的结构特征，并研究RAD51细丝形成的机制。项目4将在具有野生型和变异糖基酶的核小体的背景下重建碱基切除修复途径，并检查组蛋白一级序列变异对BER期间染色质可及性的影响。项目5将在单分子水平上定义BER和HDR酶及其变体的搜索机制。项目1-5将由Core B提供纯化蛋白，并对蛋白进行高通量分析。核心A将对所有项目进行生物信息学分析，并对项目2-4进行动力学分析

DESCRIPTION (provided by applicant): Our hypothesis is that defects in the enzyme families we study result in aberrant base excision and homology-directed repair which is an engine driving human carcinogenesis. The majority of endogenous and radiation-induced DNA lesions are removed by BER and when this pathway fails, the resulting substrates are channeled into HDR. The overall goals of this Program Project are to understand at the atomic level how three families of DNA repair enzymes, the HhH and Fpg/Nei families of DNA glycosylases and the RecA-RAD51 family of recombinases recognize and process their substrates and how germ line and tumor associated variants of these proteins influence cancer susceptibility and carcinogenesis, respectively. Based on our strengths in biochemistry and structural biology, our program is now informed and driven by the identification and characterization of germ line and tumor-associated variants of human base excision repair and HDR enzymes. Core A will identify human germ line and somatic DNA sequence variants of the oxidative DNA glycosylases and RAD51 based on structure and phylogeny. Project 1 will demonstrate whether these repair variants induce cellular transformation, are mutagenic in mouse cells and whether they influence the cellular response to ionizing radiation and chemotherapeutic agents. Project 2 will examine the biochemical properties of the oxidative glycosylase variants and solve structures of wild type enzymes with substrates and where appropriate the glycosylase variants. Project 3 will examine the biochemical and where appropriate, structural characteristics of RAD51 variants as well as study the mechanisms of RAD51 filament formation. Project 4 will reconstitute the base excision repair pathway in the context of nucleosomes with wild type and variant glycosylases and examine the effect of histone primary sequence variants on chromatin accessibility during BER. Project 5 will define at the single molecule level the search mechanisms of the BER and HDR enzymes and their variants. Projects 1-5 will be serviced by Core B which will supply purified proteins and perform high throughput analysis of the proteins. Core A will perform bioinformatics analysis for all projects and kinetics analysis for Projects 2-4