Project 2 - Structure/Function Studies of the Oxidative DNA Glycosylases

项目 2 - 氧化 DNA 糖基化酶的结构/功能研究

• 批准号: 9209396
• 负责人: Sylvie Doublie
• 金额: $ 34.53万
• 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 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9209396
• 关键词: Active Sites; Affect; Amino Acid Substitution; Bacteria; Base Excision Repairs; Binding; Biochemical; Bioinformatics; Biological; Cell Culture Techniques; Cells; Characteristics; Complex; Consultations; Crystallization; DNA; DNA Binding; DNA Damage; DNA Probes; DNA Repair; DNA Repair Enzymes; DNA glycosylase; DNA lesion; Data; Defect; Dimerization; Disease; Employee Strikes; Environment; Enzymes; Excision Repair; Exhibits; Failure; Genetic Transcription; Genomic Instability; Hand; Human; Hydantoins; Imagery; Individual; Invaded; Knowledge; Lead; Length; Lesion; Malignant Neoplasms; Maps; Methods; Molecular; Molecular Conformation; Mutation; N-terminal; NEIL3 gene; Oxides; Pathway interactions; Phenotype; Play; Point Mutation; Predisposition; Process; Protein Region; Proteins; Pyrimidines; Roentgen Rays; Role; Shapes; Single-Stranded DNA; Structure; Substrate Specificity; Time; Variant; Work; X-Ray Crystallography; base; cancer risk; cancer therapy; carcinogenesis; dimer; ds-DNA; emergency service responder; experimental study; flexibility; in vivo; insight; metaplastic cell transformation; outcome forecast; oxidative damage; programs; protein function; repaired; response; single molecule; structural biology; treatment strategy

SUMMARY There is a fundamental gap in our understanding of how mutations in enzymes of the base excision repair (BER) pathway may affect a protein's function, global conformation and its interactions with protein partners, and how these changes can lead to initiation of carcinogenesis. A powerful combination of structural, biochemical and cellular methods will be employed to study the molecular mechanisms of the human BER glycosylases that repair oxidative damage. These enzymes are the “first responders” as their task is to recognize and excise oxidized bases in DNA while leaving normal bases untouched. The central hypothesis of this program project is that defects in BER proteins can drive human carcinogenesis and affect responses to cancer treatments. The objective of Project 2 is to understand, at the biochemical and structural levels, how the BER glycosylases recognize and process oxidized lesions, how the flexible regions of the proteins influence activity and interactions with DNA or protein partners, and how single-point mutations affect the protein form and function and may ultimately initiate carcinogenesis. Guided by strong preliminary data the three aims of this proposal will 1- determine the biochemical and molecular mechanisms of lesion recognition by the NEIL glycosylases, 2- elucidate the molecular mechanisms of inhibition, activation and dimerization of NTHL1 glycosylase, and 3- evaluate the effects of BER glycosylase mutations by determining the biochemical and structural characteristics of these variants and assessing their biological phenotypes. These aims will use biochemical and structural biology methods, such as X-ray crystallography and small angle X-ray scattering (SAXS), which will be used to determine the shape and form of the full-length glycosylases and potential changes brought upon by mutations. The structure/function studies from Project 2 will work synergistically with the phenotypical characterization in human cells carried out by Project 1. Our work also dovetails with the work done in Project 3 on NTHL1 and substrate hand off, and the single-molecule studies carried out by Project 4. Core A will provide bioinformatics and statistical support for the study of the human variants. Purified proteins and human cell cultures will be provided by Core B. We anticipate that this work will provide fundamental insights into the molecular mechanisms of BER glycosylases. These results are expected to have a positive impact because they will reveal how amino acid substitutions in DNA glycosylases lead to initiation of carcinogenesis, knowledge that will be beneficial for predicting cancer susceptibility and optimizing treatment strategies.

概括 我们对碱基切除酶突变如何修复的理解存在根本差距 (BER) 途径可能影响蛋白质的功能、整体构象及其与蛋白质伙伴的相互作用， 以及这些变化如何导致癌症发生的强有力的组合。 将采用生化和细胞方法来研究人类BER的分子机制 修复氧化损伤的糖基酶这些酶是“第一响应者”，因为它们的任务是修复氧化损伤。 识别并切除 DNA 中的氧化碱基，同时保持正常碱基不变。 该项目的中心假设是 BER 蛋白的缺陷会导致人类致癌 项目 2 的目标是了解生化和癌症治疗的反应。 结构水平、BER 糖基化酶如何识别和处理氧化损伤、 蛋白质影响活性以及与 DNA 或蛋白质伴侣的相互作用，以及单点突变如何影响 影响蛋白质的形式和功能，并可能最终引发癌变。 在强有力的初步数据的指导下，该提案的三个目标将 1- 确定生化和 NEIL 糖基化酶识别病变的分子机制，2-阐明分子机制 NTHL1糖基化酶的抑制、激活和二聚化，以及3-评估BER糖基化酶的作用 通过确定这些变异的生化和结构特征并评估它们来识别突变 这些目标将使用生物化学和结构生物学方法，例如 X 射线。 晶体学和小角 X 射线散射 (SAXS)，将用于确定形状和形式 全长糖基化酶和突变带来的潜在变化的结构/功能研究。 项目 2 的研究将与人类细胞的表型表征协同工作 项目 1。我们的工作也与项目 3 中关于 NTHL1 和基板移交的工作相吻合，并且 项目4进行的单分子研究。核心A将为以下项目提供生物信息学和统计支持 纯化的蛋白质和人类细胞培养物的研究将由 Core B 提供。 我们预计这项工作将为 BER 的分子机制提供基本见解 这些结果预计将产生积极影响，因为它们将揭示氨基酸如何形成。 DNA糖基化酶的取代会导致致癌作用的发生，这一知识将有益于 预测癌症易感性并优化治疗策略。