Structural Studies of Nucleoprotein Complexes

描述（由申请人提供）：这个长期运行的项目一直专注于在DNA重组和复制中活跃的核蛋白复合物。我们一直使用的主要工具是电子显微镜和计算图像分析。在这个项目的过程中，新的和创新的方法已经开发出来，大大提高了我们理解许多不同的螺旋聚合物的结构和功能的能力。在我们对细菌RecA和人类Rad51等蛋白质的研究中发现，它们形成的聚合物是高度动态的，这些聚合物不能被简化为单一结构。相反，需要理解可能存在的状态的多样性。由于了解到诸如乳腺癌易感基因BRCA2的产物等蛋白质与Rad51相互作用并将Rad51靶向到DNA损伤位点，这些研究与疾病的相关性已经大大提高。在这项拨款的拟议扩展中，有四个目标：1)继续研究RecA/Rad51与调节这些细丝的蛋白质之间的复合物，如DinI、BRCA2、Hed1和RAD51AP1。重点将放在RecA的c端结构域和Rad51的n端结构域是如何调控的，以及调节这些丝的特性的调节蛋白如何与这些调控结构域相互作用。II)病毒单链DNA结合蛋白形成的细丝结构的测定。最初的重点将是1型单纯疱疹病毒（HSV-1） ICP8，但随后将扩展到相关的病毒蛋白，如爱泼斯坦巴尔病毒和卡波西肉瘤疱疹病毒。III)细菌偶联所需的F-pili和丝状噬菌体的结构研究。长期以来，人们一直怀疑这两者在结构上是同源的，但对这些细丝的结构研究一直非常困难。我们现在有了合适的工具，可以为两者确定相当高的分辨率结构。f -毛菌参与细菌群体内抗生素耐药性的转移，因此与公共卫生的相关性很大。形成球形病毒外壳的衣壳蛋白可以突变或修饰，从而形成螺旋管。这些管含有一种不能正确转换的蛋白质构象，因此对了解病毒组装有很大的兴趣。建议的重点是噬菌体P22、传染性法氏囊病病毒和小鼠白血病病毒的衣壳蛋白。该项目旨在了解大型蛋白质与DNA复合物的结构/功能关系。其中一些蛋白质在基因重组中是活跃的，而这些重组途径中的缺陷可能导致癌症。其他复合物涉及细菌种群内抗生素耐药性的转移，这是一个日益引起公共卫生关注的领域。其他复合物也参与了病毒的组装，包括一种导致白血病的复合物。

DESCRIPTION (provided by applicant): This long running project has been focused on nucleoprotein complexes active in DNA recombination and replication. The main tools that we have been using are electron microscopy and computational image analysis. Over the course of this project new and innovative methods have been developed that greatly increase our ability to understand the structure and function of many different helical polymers. What emerges in our studies of proteins like bacterial RecA and human Rad51 is that the polymers they form are highly dynamic, and that these polymers cannot be reduced to a single structure. Rather, the multiplicity of states that can exist needs to be understood. The relevance of these studies to disease has been greatly elevated by the understanding that proteins such as the product of the breast cancer susceptibility gene BRCA2 interact with Rad51 and target Rad51 to sites of DNA damage. In the proposed extension of this grant there are four aims: I) continued studies of complexes between RecA/Rad51 and the proteins that regulate these filaments, such as DinI, BRCA2, Hed1 and RAD51AP1. A focus will be on how the C-terminal domain of RecA and the N-terminal domain of Rad51 are regulatory, and how regulatory proteins that modulate the properties of these filaments interact with these regulatory domains. II) Determination of the structure of the filaments formed by viral single-stranded DNA binding proteins. The initial focus will be on Herpes Simplex Type 1 Virus (HSV-1) ICP8, but will then be extended to related viral proteins, such as from Epstein Barr Virus and Kaposi Sarcoma Herpes Virus. III) Structural studies of F-pili, needed for bacterial conjugation, and filamentous bacteriophage. It has long been suspected that the two are structural homologs, but structural studies of these filaments have been very difficult. We now have the tools in place to determine fairly high resolution structures for both. F-pili are involved in the transfer of antibiotic resistance within a bacterial population, so the public health relevance is great. IV) Capsid proteins that form spherical viral shells can be mutated or modified so that they form helical tubes instead. These tubes contain a conformation of the protein that fails to switch properly, and are thus of great interest in understanding virus assembly. The proposed focus is on capsid proteins from bacteriophage P22, Infectious Bursal Disease Virus, and Murine Leukemia virus. PUBLIC HEALTH RELEVANCE This project is aimed at understanding structure/function relationships in large complexes of proteins with DNA. Some of these proteins are active in genetic recombination, and defects in these recombination pathways can lead to cancer. Other complexes are involved in the transfer of antibiotic resistance within populations of bacteria, an area of growing concern for public health. And other complexes are involved in the assembly of viruses, including one that causes leukemia.