MITOCHOND NUCLEOID COMPONENT: MGM101P:BINDS & HELPS REPAIR DAMAGED MITOCHOND DNA

我们正在利用该中心的一系列技术来研究 复杂的生理过程，酵母DNA损伤反应，在一个 以系统和全面的方式来定义网络 发生在DNA复制组件之间的相互作用， 修复和细胞周期机器。我们在第二年的目标是 两个方面：第一，开发一种快速、可靠的蛋白质检测方法 允许高通量鉴定酵母的纯化 蛋白质复合体。第二，使用模型细胞过程，在此基础上 评估所采用的全球方法的有效性 并完善该中心的生成能力， 整合和传播生物数据。最后，我们有 制定了一套与 中心。我们主要选择的研究分子的方法 调节DNA损伤反应的相互作用是使用MS/MS 光谱技术用于表征蛋白质复合体的组成 用亲和层析法分离。目前，我们已经标记了 40个关键的损伤反应蛋白，并已执行 亲和-对大部分这些进行纯化。初步表征 通过MS/MS揭示了许多新的蛋白质-蛋白质相互作用。

We are exploiting the Center's array of technologies to study a complex physiological process, the yeast DNA damage response, in a systematic and comprehensive manner in order to define the network of interactions that occur among components of the DNA replication, repair, and cell cycle machineries. Our objectives in year 2 were two-fold: First, to develop a rapid and robust method of protein purification that permits high-throughput characterization of yeast protein complexes. Second, to use a model cellular process upon which to both evaluate the effectiveness of the global approaches employed at the Center and to refine the Center's ability to generate, integrate, and disseminate biological data. Finally, we have developed a working set of guidelines for collaboration with the Center. Our principal method of choice to study the molecular interactions that mediate the DNA damage response is to use MS/MS spectrometry to characterize the composition of protein complexes isolated by affinity chromatography. Currently, we have tagged over 40 key damage response proteins and have performed affinity-purification on most of these. Preliminary characterization by MS/MS has revealed a number of novel protein-protein interactions.