Structure/Function Studies of DNA Replication Initiation

描述(由申请人提供)：能够忠实地复制DNA对所有活细胞都是必不可少的。在复制之前，复制体机制必须首先由特定的复制起始因子正确地构建。广泛的研究已经确定了许多负责复制启动的蛋白质，并产生了它们的作用的一般框架。尽管如此，我们对这一过程的理解仍然存在重大差距，特别是在启动蛋白机制及其功能在生命的三个领域中被保守的程度方面。 这一建议的长期目标是阐明介导复制体组装的起始结合蛋白、复制解旋酶和解旋酶加载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，我们将研究古生菌和细菌复制启动系统。具体地说，我们的目标是：1)从生化和结构上确定古生菌CDC6/ORC1蛋白如何与特定的复制起始点相互作用；2)在原始DNA上重建二聚体和三聚体启动子复合体并对其进行生物物理表征；以及3)确定细胞内六聚体复制解旋酶的结构，该解旋酶与底物如三磷酸腺苷或特定的负载因子形成复合体。 我们已经率先对几种细菌和古生菌起始蛋白进行了结构研究。为了使我们的新工作成为可能，我们已经：1)从六种不同的生物中表达和纯化了70多种不同的全长和截断的起始因子，2)开始定义其中几种蛋白质相互作用和DNA，以及3)获得了其中一些因子的衍射数据和晶体形式。来自这些研究的信息将广泛影响一些重要的科学研究前沿，从了解分子机器的组装和功能，到提供关于新抗菌剂潜在靶标的原子分辨信息。迄今获得的数据证明了我们具体目标的可行性。

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.