PROTEIN INTERACTION NETWORKS IN DNA REPAIR

DNA 修复中的蛋白质相互作用网络

• 批准号: 6611910
• 负责人: Thomas J Begley
• 金额: $ 10.5万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT ALBANY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-07 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6611910
• 关键词: DNA repair; SDS polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; alkylation; carcinogenesis; cell cycle proteins; cell death; cytotoxicity; genetic mapping; mass spectrometry; northern blottings; phenotype; polymerase chain reaction; protein protein interaction; yeast two hybrid system

DESCRIPTION (provided by applicant) DNA damage caused by alkylating agents is recognized as an initiator of cell killing and carcinogenesis. Most cells have a similar potential to avoid these events through the coordinated action of proteins that repair DNA, yet various mammalian cell types show marked differences in viability after exposure to an alkylating agent. In the model organism Saccharomyces cerevisiae the candidate has shown that large protein networks anchored to repair pathways influence cellular viability after challenge from an alkylating agent. This suggests that accessory proteins found in a cell have prominent roles in coordinating DNA repair in vivo. Therefore, the hypothesis that human proteins that interact with key DNA repair pathways are critical for preventing alkylation induced cytotoxicity will be tested. To generate a cell specific map of protein interaction networks that influence DNA repair and gain a better understanding of factors that influence carcinogenesis and cell death, the following three aims will be pursued. First, the phenotvpe of cells that have RNA interference (RNAi) based knock-downs of important DNA repair proteins will be determined, after treatment with methyl methanesulfonate (MMS) and methylnitrosourea (MNU). This will systematically characterize the alkylation phenotype of one human cell type containing specific repair-protein deficiencies. Secondly, the pre-and post-treatment (MMS and MNU) protein-protein interactions for this group of important DNA repair proteins will be identified. Experiments will be performed using yeast two-hybrid screens and mass spectrometry based identification of protein complexes to identify networks originating from DNA repair pathways. Importantly, this will allow the identity of basal and induced protein-protein interactions. Resultant data will generate a protein map of the dynamic interactions associated with cellular responses to mutagens. Lastly, alkylation resistance networks using RNAi based phenotyping of interacting partners and DNA repair assays will be constructed. Data will be collected in a database, computationally compiled, and used to guide experiments that systematically test knock-downs for an alkylation phenotype and DNA repair activity. Collectively, results will allow: 1) to test the hypotheses, 2) generate a cell specific map of protein interaction networks in DNA repair, and 3) systematically explore the human genome for accessory proteins important for DNA repair.

描述（由申请人提供） 烷化剂引起的 DNA 损伤被认为是细胞死亡和癌变的引发剂。 大多数细胞具有相似的潜力，可以通过修复 DNA 的蛋白质的协调作用来避免这些事件，但各种哺乳动物细胞类型在暴露于烷化剂后表现出显着的活力差异。 在模型生物酿酒酵母中，候选者表明，锚定修复途径的大型蛋白质网络在受到烷化剂攻击后会影响细胞活力。 这表明细胞中发现的辅助蛋白在协调体内 DNA 修复方面发挥着重要作用。因此，与关键 DNA 修复途径相互作用的人类蛋白质对于防止烷基化诱导的细胞毒性至关重要的假设将得到检验。 为了生成影响 DNA 修复的蛋白质相互作用网络的细胞特异性图谱并更好地了解影响癌发生和细胞死亡的因素，我们将追求以下三个目标。 首先，在用甲磺酸甲酯 (MMS) 和甲基亚硝基脲 (MNU) 处理后，将确定具有基于 RNA 干扰 (RNAi) 的重要 DNA 修复蛋白敲低的细胞的表型。 这将系统地表征含有特定修复蛋白缺陷的一种人类细胞类型的烷基化表型。 其次，将确定这组重要 DNA 修复蛋白的治疗前和治疗后（MMS 和 MNU）蛋白质-蛋白质相互作用。 实验将使用酵母双杂交筛选和基于质谱的蛋白质复合物鉴定来进行，以鉴定源自 DNA 修复途径的网络。重要的是，这将允许识别基础和诱导的蛋白质-蛋白质相互作用。 所得数据将生成与细胞对诱变剂的反应相关的动态相互作用的蛋白质图。 最后，将使用基于 RNAi 的相互作用伴侣表型分析和 DNA 修复测定来构建烷基化抗性网络。 数据将被收集到数据库中，经过计算编译，并用于指导系统地测试烷基化表型和 DNA 修复活性的敲低实验。 总的来说，结果将允许：1）测试假设，2）生成 DNA 修复中蛋白质相互作用网络的细胞特异性图，3）系统地探索人类基因组中对 DNA 修复重要的辅助蛋白。