Initiation of DNA Replication of Yeast Chromosomes

描述（由申请人提供）：基因组的及时、完整和准确复制对所有真核细胞的正常增殖至关重要。过多或过少的复制都可能对细胞产生致命的后果。因此，DNA复制的起始与细胞分裂周期的进展紧密协调。该提案的总体目标是定义指导真核DNA复制起始的分子机制，并确定它们如何整合到细胞周期机制中并受其调节。为了确保复制在每个细胞周期中不会启动超过一次，真核细胞将复制启动分为两个时间上不同的事件：起点选择和起点激活。在G1期，所有潜在的复制起点都是通过复制前复合物（pre-RC）的组装来选择的。在S期，这些位点被激活，通过形成两个更高级的复合物来启动复制：前起始复合物（pre-initiation complex，pre-IC）和复制体。在前RC形成过程中，六蛋白质复制型DNA解旋酶（Mcm 2 -7复合物）以非活性状态加载到起始DNA上。前IC的形成激活Mcm 2 -7解旋酶和复制体组装所需的起始DNA的解旋酶依赖性解旋。使用生化分析，概括前RC的形成和突变体和修饰的蛋白质参与这些事件贝尔博士将：1。确定Mcm 2 -7原点加载的机制。2.确定核小体对前RC形成的影响。3.重建并解剖前IC结构。这些研究将使用酵母S。而不是人类细胞。作为DNA复制起点的高度确定的序列和强大的细胞周期工具的可用性使得在这种生物体中进行DNA复制的研究特别有利。然而，真核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翻译到人类细胞中。对复制起始事件的新理解将导致抗真菌化合物的候选靶标。此外，这些酵母研究提供的真核复制的理解将指导研究，以确定在人类中，这代表化疗药物的重要候选人相同的事件的抑制剂。在这个建议中，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数百个（如果不是数千个）启动这一过程的位点，以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增殖相关的重要信息。

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.