Base Excision Repair

项目总结(见说明)： 为了深入了解氧化DNA糖基酶是如何发生故障从而导致 基因组不稳定并导致癌症，我们建议使用结构、生化和生物学方法来 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 不仅提供了对癌症发生的基础机制的基本见解，而且还 通知人类生殖系SNP数据库，使我们能够更准确地预测哪些SNP可能 导致罹患癌症的风险增加。此外，了解人类肿瘤的变异可能会如何影响 功能将为预后和癌症治疗提供信息。具体施舍如下： 目的1，A-解决人类FPG/Nei家族成员NEIL1、NEIL2和 NEIL3，或接近的同源物，如与其DNA底物形成复合体的模拟病毒Nei蛋白。朝向 为了实现这个目标，我们已经获得了MimiVirus Neil与胸腺嘧啶二醇和胸腺嘧啶二醇的配合物。 螺亚氨基二海因，这是第一个与氧化DNA形成复合体的Nei酶的例子 损伤。一旦用结合口袋中的底物确定了结构，定点突变体将 为进一步的生化分析而建造。C-检验NEILI、2和3变种的假设 在项目1和核心A中确定的通过确定生化特征来促进癌症的发生 这些变体的底物专一性和DNA结合特性，以及它们的晶体 结构。 目标2.A-解决人类第N个超家族成员或近亲直系物的晶体结构 与它们的DNA底物形成复合体。值得注意的是，目前还没有人能够将hNTHI具体化。B-to 检查在项目1和核心A中确定的一组精选的hNTHI生殖系变体 生化和结构分析确定哪些变异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中的生物学研究提供信息，并提供对 项目4中糖基酶与核小体的相互作用、纯化蛋白和快速酶分析 将由Core B提供。Core 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,