Initiation of DNA Replication of Yeast Chromosomes

描述(申请人提供)：基因组的及时、完整和准确复制对于所有真核细胞的正常增殖是必不可少的。复制过多或过少都会对细胞造成致命的后果。因此，DNA复制的启动与细胞分裂周期的进展密切相关。这项建议的总体目标是定义指导真核DNA复制启动的分子机制，并确定它们如何整合到细胞周期机制中并受其调控。为了确保每个细胞周期的复制不超过一次，真核细胞将复制开始分为两个时间上不同的事件：起始点选择和起始点激活。在G1期，所有潜在的复制起点都是通过组装复制前复合体(Pre-RCs)来选择的。在S期，这些位点被激活，通过形成两个更高级别的复合体来启动复制：预起始复合体(Pre-IC)和复制体。在前RC形成过程中，由六个蛋白质组成的复制DNA解旋酶(McM2-7复合体)以不活跃的状态被加载到原始DNA上。前IC的形成激活了McM2-7解旋酶和复制体组装所需的依赖解旋酶的原始DNA的解离。利用生化分析来概括前RC的形成以及参与这些事件的突变和修饰蛋白，贝尔博士将：1.确定McM2-7起源负荷的机制。2.确定核小体对前RC形成的影响。3.重建和解剖前冰期地层。这些研究将使用酿酒酵母而不是人类细胞进行。作为DNA复制起点的高度定义的序列的可用性和强大的细胞周期工具使得对这种生物中的DNA复制的研究特别有利。然而，真核细胞DNA复制的高度保守性表明，在这种酵母中取得的进展将迅速转化为人类细胞。对复制启动事件的新理解将导致抗真菌化合物的候选靶点。此外，这些酵母研究提供的对真核复制的理解将指导研究，以确定人类中相同事件的抑制剂，这些抑制剂代表了化疗药物的重要候选药物。项目叙述在这项提案中，贝尔博士将研究控制动物细胞染色体复制的基本事件。特别是，他将研究如何选择和激活启动这一过程的数百个(如果不是数千个)位置，以便在适当的时间开始染色体复制。这些发现将提供与包括人类在内的所有动物细胞的增殖相关的基本重要信息。

DESCRIPTION (provided by applicant): The timely complete and accurate replication of the genome is essential to the normal proliferation of all eukaryotic cells. Either too much or too little replication can have lethal consequences for the cell. Accordingly, the initiation of DNA replication is tightly coordinated with progression through the cell division cycle. The overall goal of this proposal is to define the molecular mechanisms that direct the initiation of eukaryotic DNA replication and determine how they are integrated into and regulated by the cell cycle machinery. To ensure that replication does not initiate more than once per cell cycle, eukaryotic cells divide the replication initiation into two temporally distinct events: origin selection and origin activation. During G1-phase, all potential origins of replication are selected through the assembly of pre-replication complexes (pre-RCs). During S-phase, these sites are activated to initiate replication by the formation of two higher order complexes: the pre-initiation complex (pre-IC) and the replisome. During pre-RC formation the six-protein, replicative DNA helicase (the Mcm2-7 complex) is loaded onto origin DNA in an inactive state. Formation of the pre-IC activates the Mcm2-7 helicase and the helicase-dependent unwinding of origin DNA that is required for replisome assembly. Using biochemical assays that recapitulate pre-RC formation and mutant and modified proteins involved in these events Dr. Bell will: 1. Determine the mechanism of Mcm2-7 origin loading. 2. Determine the impact of nucleosomes on pre-RC formation. 3. Reconstitute and dissect pre-IC formation. These studies will be performed using the yeast S. cerevisiae rather than human cells. The availability of highly defined sequences that act as origins of DNA replication and powerful cell cycle tools make studies of DNA replication in this organism particularly advantageous. Nevertheless, the highly conserved nature of eukaryotic DNA replication indicates that progress made in this yeast will be translated rapidly to human cells. New understanding of the events of replication initiation will lead to candidate targets for anti-fungal compounds. Moreover, the understanding eukaryotic replication provided by these yeast studies will direct studies to identify inhibitors of the same events in humans, which represent important candidates for chemotherapeutic agents. Project Narrative In this proposal Dr. Bell will study the fundamental events that control the duplication of animal cell chromosomes. In particular, he will investigate how the hundreds, if not thousands, of sites at which this process is initiated are selected and activated to start chromosome duplication at the appropriate times. The findings will provide fundamentally important information that will be relevant to the proliferation of all animal cells including humans.