Structure and Function of DNA Repair Enzymes

我们的假设是，我们研究的酶家族中的缺陷导致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损伤通过碱基切除修复（BER）机制去除，并且当该途径失败时，所产生的底物被引导进入同源定向修复。该计划项目的总体目标是在原子水平上了解三个DNA修复酶家族（DNA糖基化酶的HhH-GPD超家族、DNA糖基化酶的Fpg/Nei家族和重组酶的RecA-RAD 51家族）如何识别和处理其底物，以及这些蛋白质的生殖系和肿瘤相关变体如何分别影响癌症易感性和致癌作用。为了将我们的基础科学更直接地转化为癌症，我们现在建议利用我们的专业知识和经过测试的方法来检查人类遗传变异。基于我们对新型底物特异性和生物化学活性的发现，以及我们在基础生物化学和结构生物学方面的优势，我们的更新计划将通过对人类碱基切除修复和同源定向修复酶的种系和肿瘤相关变体的鉴定和表征来了解和推动。核心A将根据结构和同源性鉴定氧化DNA糖基化酶和RAD 51的人生殖系和体细胞DNA序列变体。项目1将证明这些修复变体是否诱导细胞转化，在小鼠细胞中是否具有致突变性，以及它们是否影响细胞对电离辐射和化疗剂的反应。项目2将研究氧化糖基化酶变体的生化特性，并解决野生型酶与底物的结构，并在适当的情况下解决糖基化酶变体的结构。项目3将研究RAD 51变体的生物化学和适当的结构特征，以及研究RAD 51细丝形成的机制。项目4将在具有野生型和变体糖基化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体对BER期间染色质可及性的影响。项目1-4将由蛋白质和生物化学核心B提供服务，该核心将提供纯化蛋白质并对蛋白质进行高通量分析。除了所有项目的生物信息学外，核心A还将为项目2-4进行动力学分析。核心C将为该项目提供行政支持。

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 the base excision repair (BER) machinery and when this pathway fails, the resulting substrates are channeled into homology-directed repair. The overall goals of this Program Project are to understand at the atomic level how three families of DNA repair enzymes the HhH-GPD superfamily of DNA glycosylases, the Fpg/Nei family 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. In order to translate our basic science more directly to cancer, we now propose to use our expertise and tested methodologies to examine human genetic variation. Based on our discoveries of novel substrate specificities and biochemical activities, as well as our strengths in fundamental biochemistry and structural biology, our program for the renewal will be informed and driven by the identification and characterization of germ line and tumor-associated variants of human base excision repair and homology-directed repair 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. Projects 1-4 will be serviced by the Protein and Biochemistry Core B which will supply purified proteins and perform high throughput analysis of the proteins. In addition to bioinformatics for all projects, Core A will also perform kinetics analysis for Projects 2-4. Core C will provide the administrative underpinnings for the project.