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 我们对基本惊喜修复酶中突变的理解有根本的差距 （BER）途径可能会影响蛋白质功能，全球会议及其与蛋白质伴侣的相互作用， 以及这些变化如何导致癌变的主动性。结构的强大组合， 将采用生化和细胞方法来研究人类BER的分子机制 修复氧化物损伤的糖基酶。这些酶是“第一响应者”，因为他们的任务是 在DNA中公认并经历了氧化的碱，同时使正常碱不受欢迎。 该计划项目的中心假设是，BER蛋白的缺陷可以驱动人类致癌 并影响对癌症治疗的反应。项目2的目的是在生化和 结构水平，BER糖基酶如何识别和处理氧化病变，如何柔性区域 蛋白质会影响活性和与DNA或蛋白质伴侣的相互作用，以及单点突变 影响蛋白质形式和功能，并最终可能引发致癌作用。 在强大的初步数据的指导下，该提案的三个目标将确定生化和 尼尔糖基化酶识别病变的分子机制，2阐明了分子机制 抑制作用，NTHL1糖基化酶的激活和二聚化，并评估BER糖基化酶的作用 通过确定这些变体的生化和结构特征并评估它们的突变 生物表型。这些目标将使用生化和结构生物学方法，例如X射线 晶体学和小角度X射线散射（SAXS），将用于确定形状和形式 突变带来的全长糖基酶和潜在的变化。结构/功能研究 来自项目2将与人类细胞中的表型表征协同作用 项目1。我们的工作也与NTHL1和基板上的项目3中完成的工作相吻合， 项目4进行的单分子研究。核心A将为生物信息学和统计支持提供 人类变体的研究。纯化的蛋白质和人类细胞培养物将由核心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 糖基酶。预计这些结果将产生积极的影响，因为它们将揭示氨基酸的方式 在DNA糖基酶中取代导致癌变的开始，这将有益于 预测癌症的敏感性并优化治疗策略。