Structural Studies of Nucleoprotein Complexes

核蛋白复合物的结构研究

• 批准号: 6635928
• 负责人: EDWARD H. EGELMAN
• 金额: $ 30.34万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-04-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6635928
• 关键词: DNA binding protein; DNA repair; adenosine triphosphate; automated data processing; bacterial proteins; binding sites; computer simulation; conformation; cryoelectron microscopy; enzyme substrate complex; genetic recombination; helicase; intermolecular interaction; model design /development; nuclear magnetic resonance spectroscopy; nucleic acids; nucleoproteins; physical model; polymers; protein folding; protein structure function; recombinase; simian virus 40; statistics /biometry; structural biology; tumor antigens

DESCRIPTION (provided by applicant): Much of the research on homologous recombination has been focused on the bacterial RecA protein. The eukaryotic Rad51 protein forms very similar nucleoprotein filaments to those formed by RecA. Rad51 is essential in higher organisms, including humans, and is believed to play a large role in the maintenance of genome stability. Electron microscopic (EM) studies of these protein-DNA filaments can yield important information about mechanism in these highly conserved structures. Significant improvements in resolution are now possible using cryo-EM of frozen-hydrated specimens, as well as new computational approaches. These studies are beginning to show how domains are arranged in these filaments, the conformational changes that are associated with activation of the filaments, as well as revealing interactions between these filaments and other proteins. Helicases have the same nucleotide-binding core present in the RecA and Rad5 I proteins. It has become apparent that a large number of helicases involved in DNA recombination, replication, repair and transcription form hexameric rings around DNA. Mutations in helicase genes have been shown in humans to lead to xeroderma pigrnentosa, Cockayne's syndrome, Werner's syndrome and Bloom's syndrome. There are numerous suggestions that many of these proteins may not actually be helicases, but motor proteins with diverse functions. In fact, more than 2 percent of human genes may encode for "helicases." EM and single particle image analysis will be used on different hexameric replicative helicases, including E. coli DnaB, Simian Virus 40 large T, archaeal MCM and bacteriophage T7 gp4, to study the interaction with DNA, conformational changes that occur during the ATPase cycle, and cooperative interactions among subunits. These studies will be done in collaboration with crystallographic efforts, and high-resolution data on subunits and domains will be combined with lower resolution data on quatemary organization. The overall question that will be addressed is whether the conserved nucleotide-binding core present in RecA, Rad5 1 and the helicase superfamily has led to a conservation of mechanism across this huge class of proteins. Structural studies are now revealing that other proteins active in recombination and repair, with no apparent homology to this class, also form rings. Examples are translin, involved in chromosomal translocation and RNA processing, and Rad52. Studies of these proteins will be conducted in parallel, with the aim of understanding whether convergent mechanisms of protein-mediated recombination and repair exist.

描述（由申请人提供）：关于同源的大部分研究 重组已集中在细菌RECA蛋白上。真核生物 RAD51蛋白与由核蛋白丝形成非常相似的核蛋白丝与 reca。 RAD51在包括人在内的高等生物中至关重要，并且被认为是必不可少的 在维持基因组稳定性中发挥重要作用。电子 这些蛋白-DNA丝的显微镜（EM）研究可以产生重要的 这些高度保守的结构中有关机制的信息。重要的 现在，使用冷冻水合的冷冻EM可以改善分辨率 标本以及新的计算方法。这些研究已经开始 为了显示这些细丝中的域如何布置，构象变化 与细丝的激活相关的以及揭示 这些细丝与其他蛋白质之间的相互作用。解旋酶具有 RECA和RAD5 I蛋白中存在相同的核苷酸结合核心。它有 显然，大量参与DNA重组的解旋酶， 在DNA周围复制，修复和转录形成六聚环。 在人类中已显示出解旋酶基因的突变，导致静脉 Pigrnentosa，Cockayne's综合征，Werner's综合征和Bloom综合征。那里 有许多建议，这些蛋白质中的许多实际上可能不是 解旋酶，但具有不同功能的运动蛋白。实际上，超过2个 人类基因的百分比可能编码为“解旋酶”。 EM和单个粒子图像 分析将用于不同的六聚体复制式解旋酶，包括 大肠杆菌DNAB，Simian病毒40大T，古细菌MCM和噬菌体T7 GP4， 为了研究与DNA的相互作用，在此期间发生的构象变化 ATPase周期和亚基之间的合作相互作用。这些研究会 可以与晶体学工作和高分辨率合作完成 亚基和域的数据将与较低的分辨率数据相结合 Quatemary组织。 将要解决的总体问题是保守 RECA，RAD5 1中存在的核苷酸结合核心和解旋酶超家族 导致了这种巨大类蛋白质的机制的保护。 结构研究现在揭示了其他蛋白质活跃 重组和维修，与此类没有明显同源，也形成 戒指。例子是翻译，参与染色体易位和RNA 加工和RAD52。这些蛋白质的研究将并行进行 目的是了解蛋白质介导的收敛机理 存在重组和修复。