Replication Initiation in Bacteria and Eukaryotes

描述(由申请人提供)：这项工作的目标是对DNA复制是如何启动的基于结构的理解。复制始于基因组上的多个位置，称为DNA复制起点。专门的蛋白质复合体结合在这些位点上，并为复制准备双链。这些集合的活动受到严格控制，以确保每个细胞周期只复制一个基因组。这些大型复合体的结构、调节和作用机制尚不完全清楚。我们的工作具有实际意义，因为起源复合体的调控变化有助于人类癌症的发生。细菌复合体的努力将为开发新的抗生素提供亟需的靶标。我们研究真核生物和细菌中的复制起始复合体。大量的工作描述了起源复合体的组成部分以及它们如何运作的细节。然而，在这些情况下，缺少的是启动机器的三维视图，以指导我们理解这些组件如何协同工作。我们研究的总体目标就是提供这样的观点。目前的提案结合了三种互补的方法，侧重于原产地识别和DNA解链所涉及的几个组成部分。目的#1针对几个细菌螺旋体复合体，它们的结构和功能可以在复杂的水平上被解剖，并将有助于我们对真核复合体的理解。第二个目标是获得和分析完整的ORC系综。目的#3利用我们最近发现的一个独特的细菌MCM复合体，它更容易处理，但仍然应该为其真核同行提供一个忠实的模型。与公共健康相关的细胞通过在基因组上被称为DNA复制起点的位置上沉积大分子组件来为下一轮DNA复制做准备。新出现的证据表明，这些DNA复制起始组件代表了设计新疗法所针对的丰富分子靶点的集合。在发现抗癌药物的努力中，这一途径并没有得到很好的代表。缺乏有关相关分子靶点的有用结构信息阻碍了结构导向药物设计的努力。我们的结构确定工作将推动发现新的抗癌药物的努力。此外，我们在细菌复合体方面的工作将为开发新的抗生素提供急需的目标。

DESCRIPTION (provided by applicant): The goal of this work is a structure-based understanding of how DNA replication is initiated. Replication initiates at multiple sites on the genome called origins of DNA replication. Specialized protein complexes bind at these sites and prepare the duplex for replication. The activity of these ensembles is tightly controlled to ensure that only one copy of the genome is made per cell cycle. The architecture, regulation and mechanisms of action of these large complexes are incompletely understood. Our work is of practical significance because regulatory changes in origin complexes contribute to human cancers. Efforts with bacterial complexes will provide much needed targets for development of novel antibiotics. We study replication initiation complexes in eukaryotes and bacteria. A large body of work has described components of origin complexes and details of how they operate. What is missing, however, in any of these cases, is a three-dimensional view of the initiation machine to guide our understanding of how these components work together. The overall goal of our research is to provide such views. The current proposal incorporates three complementary approaches that focus on several components involved in origin recognition and DNA unwinding. Aim #1 is directed at several bacterial helicaseloader complexes, whose structure and function can be dissected at a sophisticated level and will inform our understanding of eukaryotic complexes. Aim #2 is addressed at obtaining and analyzing the complete ORC ensemble. Aim #3 exploits our recent identification of a unique bacterial MCM complex, which is more tractable, but should still provide a faithful model for its eukaryotic counterparts. PUBLIC HEALTH RELEVANCE Cells prepare for the next round of DNA replication by depositing macromolecular assemblies onto sites on the genome termed origins of DNA replication. Emerging evidence suggests that these DNA replication initiation assemblies represent a rich collection of molecular targets against which to design novel therapeutics. This pathway is not well represented in efforts to discover anti-cancer agents. Lack of useful structural information on relevant molecular targets has stymied efforts at structure-guided drug design. Our structure determination efforts will advance efforts to discover novel anti-cancer agents. Also, our work with bacterial complexes will provide much needed targets for development of novel antibiotics.