Structure/Function Studies of DNA Replication Initiation

描述（由申请人提供）：忠实复制DNA的能力对所有活细胞都至关重要。在复制开始之前，复制体机器必须首先由特定的复制起始因子正确构建。广泛的研究已经确定了许多负责复制起始的蛋白质，并为它们的作用提供了一个总体框架。尽管如此，我们对这一过程的理解仍然存在重大差距，特别是在起始蛋白机制及其功能在生命的三个领域中保守的程度方面。 这个建议的长期目标是阐明几个关键的结构/功能关系的起源结合起始蛋白，复制解旋酶，解旋酶加载因子介导的复制体组装。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的复制起始系统。具体而言，我们的目标是：1）在生物化学和结构上确定古细菌Cdc 6/Orc 1蛋白如何与特定复制起始位点相互作用，2）在起始DNA上重建二聚体和三聚体起始物复合物并在生物化学上表征，以及3）确定与底物如ATP复合或与专门的加载因子复合的细胞六聚体复制解旋酶的结构。 我们已经率先对几种细菌和古细菌起始蛋白进行了结构研究。为了使我们新提出的努力，我们已经：1）表达和纯化了来自六种不同生物的70多种不同的全长和截短的起始因子，2）开始确定这些蛋白质中的几种蛋白质彼此之间和DNA之间的相互作用，3）获得了这些因子中的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许多重要的科学研究前沿，从理解分子机器的组装和功能，到为新抗菌剂的潜在靶点提供原子分辨率信息。迄今为止获得的数据表明，我们的具体目标是可行的。

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.