Base Excision Repair

项目总结（见说明）： 为了深入了解氧化DNA糖基化酶如何可能发生故障，从而导致 基因组不稳定和导致癌症，我们建议使用结构，生化和生物学方法， 阐明这些酶如何执行碱基切除修复的第一步。这个项目的长期目标是 不仅提供了对致癌机制的基本见解， 告知人类生殖系SNP数据库，并允许我们更准确地预测哪些SNP可能 导致癌症风险增加。此外，了解人类肿瘤中的变异如何影响 功能将告知预后和癌症治疗。具体布施如下： 目的1，A-为了解析Fpg/Nei家族NEIL 1、NEIL 2和NEIL 3的人类成员的晶体结构， NEIL 3，或接近的直系同源物，如与其DNA底物复合的拟病毒Nei蛋白。朝向 为此，我们已经获得了拟病毒尼尔与胸腺嘧啶乙二醇的复合物， 螺亚氨基二乙内酰脲，这是与氧化DNA复合的任何Nei酶的第一个例子 损伤。B-一旦结合口袋中的底物确定了结构，定点突变体将 用于进一步的生化分析。C-检验NEILI、2和3变体 在项目1和核心A中确定的通过确定生物化学特征促进致癌作用 这些变体的特性，如底物特异性和DNA结合特性，以及它们的晶体 结构. 目标2. A-解析第N超家族的人类成员的晶体结构或 与它们的DNA底物复合。值得注意的是，还没有人能够结晶hNTHI。B-至 检查在项目1和核心A中鉴定的一组选择的hNTHI种系变体， 生物化学和结构分析确定哪些变体可能具有潜在的有害作用， 后果 项目2的结构/功能研究将为项目1的生物学研究提供信息，并提供以下方面的见解 糖基化酶与核小体的相互作用 将由核心B提供。核心A将帮助设计和分析酶动力学实验，

PROJECT SUMMARY (See Instructions): In order to provide insight into how the oxidative DNA glycosylases may malfunction and thus lead to genome instability and cause cancer, we propose to use structural, biochemical and biological approaches to elucidate how these enzymes perform the first step In base excision repair. The long-term goal of this project is to not only provide fundamental insights into the mechanisms underpinning carcinogenesis but also to inform the human germ line SNP databases and allow us to predict with greater accuracy which SNP may lead to an increased risk of cancer. Moreover, understanding how variants In human tumors may affect function will inform prognosis and cancer treatment. The specific alms are as follows: Aim 1, A- To solve the crystal structures of the human members of the Fpg/Nei family NEIL1, NEIL2, and NEIL3, or close orthologs such as the mimivirus Nei proteins in complex with their DNA substrates. Towards this goal we have already obtained complexes of Mimivirus Neil with thymine glycol and spiroiminodihydantoin, which are the first examples of any Nei enzyme in complex with an oxidative DNA lesion. B- Once the structures are determined with substrate in the binding pocket, site directed mutants will be constructed for further biochemical analyses. C- To test the hypothesis that the NEILI, 2 and 3 variants Identified In Project 1 and Core A contribute to carcinogenesis by determining the biochemical characteristics of these variants such as substrate specificity and DNA binding properties and, where Indicated, their crystal structures. Aim 2. A- To solve the crystal structures of human members of the Nth superfamily or close orthologs in complex with their DNA substrates. Of note Is that no one has yet been able to crystallize hNTHI. B- To examine a select group of hNTHI germ line variants identified in Project 1 and Core A and through biochemical and structural analysis determine which variants may have potentially deleterious consequences. Structure/function studies from Project 2 will Inform the biological studies in Project 1 and provide insight into the interactions of glycosylases with nucleosomes In Project 4, Purified proteins and rapid enzyme analysis will be provided by Core B. Core A will help design and analyze enzyme kinetics experiments,