Structure and Function of DNA Repair Enzymes

DNA修复酶的结构和功能

• 批准号: 8327279
• 负责人: SUSAN S. WALLACE
• 金额: $ 196.78万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-03 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8327279
• 关键词: Affect; Amino Acid Substitution; Automobile Driving; Base Excision Repairs; Basic Science; Biochemical; Biochemistry; Bioinformatics; Biological; Cancer Biology; Cancer Etiology; Cells; Characteristics; Chromatin; Chromatin Structure; Clinical; DNA; DNA Damage; DNA Repair; DNA Repair Enzymes; DNA Repair Gene; DNA Repair Pathway; DNA Sequence; DNA glycosylase; DNA lesion; Databases; Defect; Environment; Enzyme Kinetics; Enzymes; Event; Excision; Exhibits; Exposure to; Family; Family Study; Family member; Filament; Funding; Future; Genes; Genetic; Genetic Variation; Genomic Instability; Germ Lines; Goals; Histones; Human; Human Genetics; Individual; Interdisciplinary Study; Internet; Ionizing radiation; Kinetics; Knowledge; Laboratories; Lesion; Light; Malignant Neoplasms; Measurement; Measures; Metabolism; Methodology; Methods; Molecular; Mus; Nucleosomes; Oncogenic; Organism; Pathway interactions; Phase; Phylogeny; Population; Positioning Attribute; Predisposition; Principal Investigator; Process; Property; Protein Biochemistry; Proteins; Publishing; Radiation; Recruitment Activity; Role; Services; Site; Specificity; Structural Biologist; Structure; Substrate Specificity; System; Testing; The Cancer Genome Atlas; Therapeutic; Translating; Ursidae Family; Variant; Work; base; cancer risk; cancer therapy; carcinogenesis; chemotherapeutic agent; design; high throughput analysis; human cancer mouse model; insight; metaplastic cell transformation; mutant; neoplastic cell; novel; novel strategies; programs; recombinase; reconstitution; repair enzyme; repaired; research study; response; structural biology; tumor; tumor progression

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.

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损伤都是通过碱基切除修复（BER）机制去除的，当这一途径失败时，产生的底物被引导到同源定向修复中。本项目的总体目标是在原子水平上了解三个DNA修复酶家族（HhH-GPD DNA糖基酶超家族、Fpg/Nei DNA糖基酶家族和RecA-RAD51重组酶家族）如何识别和处理它们的底物，以及这些蛋白的种系和肿瘤相关变体如何分别影响癌症易感性和癌变。为了将我们的基础科学更直接地转化为癌症，我们现在建议使用我们的专业知识和经过测试的方法来检查人类基因变异。基于我们对新的底物特异性和生化活性的发现，以及我们在基础生物化学和结构生物学方面的优势，我们的更新计划将由人类碱基切除修复和同源导向修复酶的生殖系和肿瘤相关变体的鉴定和表征所决定和驱动。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1-4将由蛋白质和生物化学核心B提供服务，该核心B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，Core A还将对项目2-4进行动力学分析。核心C将为该项目提供管理基础。