Structure/Function Studies of DNA Replication Initiation

DNA复制起始的结构/功能研究

• 批准号: 7060375
• 负责人: JAMES M BERGER
• 金额: $ 25.51万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7060375
• 关键词: Archaea; DNA binding protein; DNA replication; DNA replication origin; SDS polyacrylamide gel electrophoresis; X ray crystallography; bacterial proteins; crystallization; enzyme activity; helicase; high performance liquid chromatography; ion exchange chromatography; nucleic acid structure; polymerase chain reaction; protein protein interaction; protein purification; protein structure function; Archaea; DNA binding protein; DNA replication; DNA replication origin; SDS polyacrylamide gel electrophoresis; X ray crystallography; bacterial proteins; crystallization; enzyme activity; helicase; high performance liquid chromatography; ion exchange chromatography; nucleic acid structure; polymerase chain reaction; protein protein interaction; protein purification; protein structure function

DESCRIPTION (provided by applicant): The ability to faithfully replicate DNA is essential to all living cells. Before replication can ensue, replisomal machineries must first be properly constructed by specific replication initiation factors. Extensive studies have identified many of the proteins responsible for replication initiation and have produced a general framework for their action. Nonetheless, significant gaps remain in our understanding of this process, particularly, with respect to initiation protein mechanisms and the degree to which their function is conserved across the three domains of life. The long-term objective of this proposal is to illuminate several key structure/function relationships of the origin-binding initiator proteins, replicative helicases, and helicase-loader factors that mediate replisome assembly. To compare and contrast the function of these proteins in different organisms, we will study archaeal and bacterial replication initiation systems. Specifically, we aim to: 1) biochemically and structurally determine how the archaeal Cdc6/Orc1 protein interacts with specific replication origin sites, 2) reconstitute and biophysically characterize dimeric and trimeric initiator complexes on origin DNAs, and 3) determine the structure of a cellular, hexameric replicative helicase, complexed either with substrates such as ATP, or with a specialized loading factor. We have already pioneered structural studies of several bacterial and archaeal initiation proteins. To enable our new proposed efforts, we have: 1) expressed and purified over 70 different full-length and truncated initiation factors from six different organisms, 2) begun to define the interaction of several of these proteins with each other and DNA, and 3) obtained diffraction data and crystal forms for some of these factors. Information from these studies will broadly impact a number of important scientific research fronts, from understanding the assembly and function of molecular machines, to providing atomic resolution information on potential targets for new antimicrobials. Data obtained to date demonstrate the feasibility of our specific aims.

描述（由申请人提供）：忠实复制DNA的能力对于所有活细胞都是必不可少的。在进行复制之前，必须首先通过特定的复制启动因子正确构建重新构造机械。广泛的研究已经确定了许多负责复制启动的蛋白质，并为其作用产生了一般框架。尽管如此，我们对这一过程的理解仍然存在，特别是在起始蛋白机制以及其在生命三个领域中保守其功能的程度。 该提案的长期目标是阐明原点结合起始蛋白，复制型解旋酶和介导介导重壳体组装的动力酶载体因子的几个关键结构/功能关系。为了比较和对比这些蛋白质在不同生物体中的功能，我们将研究古细菌和细菌复制起始系统。具体而言，我们的目的是：1）生化和结构上确定古细菌CDC6/ORC1蛋白如何与特定复制起源位点相互作用，2）重新构建和生物物理表征二聚体和三聚体在原点DNA上的复合体，以及3），以及与A细胞复制的结构，同类螺旋酶的结构，以及与分散的螺旋构成或复杂的载体或相同的载体或相同的载体或类似的载体。 我们已经开创了几种细菌和古细菌起始蛋白的结构研究。为了实现我们的新提议的努力，我们有：1）从六种不同生物体中表达并纯化了70种不同的全长和截断起始因子，2）开始定义这些蛋白质中的几种相互作用，而DNA以及3）获得了这些因素的衍射数据和晶体形式。这些研究的信息将广泛影响许多重要的科学研究方面，从了解分子机器的组装和功能到提供有关新抗微生物剂的潜在靶标的原子分辨率信息。迄今为止获得的数据证明了我们特定目标的可行性。