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

总结 我们对碱基切除修复酶突变的理解存在根本性的空白 (BER)信号通路可能影响蛋白质的功能、整体构象及其与蛋白质伴侣的相互作用， 以及这些变化如何导致致癌作用的开始。一个强大的组合结构， 生物化学和细胞学方法将被用来研究人类BER的分子机制 修复氧化损伤的糖基化酶。这些酶是“第一反应者”，因为它们的任务是 识别并切除DNA中的氧化碱基，而不影响正常碱基。 该项目的中心假设是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的目标是了解，在生物化学和 结构水平，BER糖基化酶如何识别和处理氧化损伤， 这些蛋白质影响DNA或蛋白质伴侣的活性和相互作用，以及单点突变 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，本提案的三个目标将是：1-确定生物化学， NEIL糖基化酶识别病变的分子机制，2-阐明分子机制 NTHL 1糖基化酶的抑制、活化和二聚化，以及3-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其 生物表型这些目标将使用生物化学和结构生物学方法，如X射线 晶体学和小角X射线散射（SAXS），这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。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。我们的工作还与项目3中关于NTHL 1和基板交接的工作相吻合， 项目4进行的单分子研究。核心A将提供生物信息学和统计支持， 人类变异的研究。纯化蛋白和人细胞培养物将由核心B提供。 我们期望这项工作将为BER的分子机制提供基本的见解 糖基化酶这些结果预计将产生积极的影响，因为它们将揭示氨基酸 DNA糖基化酶中的取代导致致癌作用的启动，这一知识将有利于 预测癌症易感性和优化治疗策略。

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.