NMR Studies of Ku70 and Ku80 Domains

描述(由申请者提供)：蛋白质-DNA识别是许多 生物过程。这种交互可以分为两种主要类型： 特定于序列和特定于结构。DNA修复蛋白通常不会 识别特定的DNA序列，但搜索不寻常的DNA结构 需要维修。此外，DNA修复过程通常需要 几种蛋白质的协同作用。我们对结构的理解 DNA修复过程的生物学仍处于初级阶段。一些主要的DNA 修复途径还没有在分子水平上得到表征。这个 结构特异性DNA识别机制及其相互作用 修复复合体中蛋白质之间的相互作用还不是很清楚。 在这项提案中，我们计划描述双链的主要途径 DNA断裂修复。双链DNA断裂是由电离辐射引起的 (D)J重组和生理氧化反应，并适当修复 对维持基因组稳定性很重要。这些蛋白质对细胞内的 双链断裂修复的主要途径是Ku70(69.8kD)和Ku80 (82.7KD)。这两种蛋白质形成紧密的复合体，结合到断裂的DNA末端，并 招募其他蛋白质，包括DNA依赖的蛋白激酶和 DNA连接酶形成DNA修复机制。先前的研究表明， Ku蛋白含有结构和功能独立的结构域。我们会 分析了Ku结构域的结构，并对其形成机制进行了研究。 这些结构域之间的相互作用，以及它们与DNA和其他 DNA修复机制中的蛋白质使用核磁的组合 核磁共振和分子生物学方法。这些研究将提供 在分子水平上，对蛋白质在 DNA双链断裂部位。这些信息对于改进我们的 对Ku介导的DNA双链断裂分子机制的认识 修复途径。这项研究也应该提高我们对 DNA修复的一般结构生物学。

DESCRIPTION (provided by applicant): Protein-DNA recognition is central to many biological processes. This interaction can be classified into two major types: sequence-specific and structure-specific. DNA-repair proteins generally do not recognize specific DNA sequences but search for unusual DNA structures which require repair. In addition, the DNA repair process often requires the concerted actions of several proteins. Our understanding of the structural biology of DNA-repair process is still in its infancy. A number of major DNA repair pathways have not been characterized at the molecular level. The mechanism of structure-specific DNA recognition as well as the interactions between proteins in the repair complex is not well understood. In this proposal, we plan to characterize the major pathway for double-strand DNA breaks repair. Double-strand DNA breaks are caused by ionizing radiation, V (D) J recombination and physiological oxidation reactions, and proper repair is important to maintain genomic stability. The proteins that are central to the major pathway of double-strand-break repair process are Ku70 (69.8 kD) and Ku80 (82.7 kD). These two proteins form a tight complex, bind to broken DNA ends and recruit other proteins, including a DNA-dependent protein kinase and a DNA-ligase to form the DNA repair machinery. Previous studies suggest that the Ku proteins contain structurally and functionally independent domains. We will analyze the structures of Ku domains and study the mechanism of the interactions between these domains, and their interactions with DNA and other proteins in the DNA-repair machinery using a combination of nuclear magnetic resonance (NMR) and molecular biological approaches. These studies will provide an understanding, at the molecular level, of the assembly of the proteins at DNA double strand break sites. This information is necessary to improve our knowledge of the molecular mechanism of the Ku-mediated DNA double-strand break repair pathway. This study should also improve our understanding of the structural biology of DNA repair in general.