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。复制过多或过少都会对细胞造成致命的后果。因此，DNA复制的开始与细胞分裂周期的进展密切协调。本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。为了确保每个细胞周期内复制不会启动一次以上，真核细胞将复制启动分为两个在时间上不同的事件：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预复制复合体（pre-RCs）的组装来选择的。在s期，这些位点被激活，通过形成两个高级复合体来启动复制：起始前复合体（pre-initiation complex, pre-IC）和复制体。在pre-RC形成过程中，六蛋白复制DNA解旋酶（Mcm2-7复合体）在非活性状态下被装载到起始DNA上。pre-IC的形成激活了Mcm2-7解旋酶和起源DNA的解旋酶依赖性解旋，这是复制体组装所必需的。Bell博士将使用生化分析来概括这些事件中涉及的pre-RC形成以及突变和修饰的蛋白质：确定Mcm2-7原点加载机理。2. 确定核小体对预rc形成的影响。3. 重建和解剖前ic形成。这些研究将使用酿酒酵母而不是人类细胞进行。作为DNA复制起源的高度定义的序列的可用性和强大的细胞周期工具使得研究这种生物的DNA复制特别有利。然而，真核生物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。对复制起始事件的新认识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确定人类相同事件的抑制剂，这些抑制剂代表了化疗药物的重要候选者。在这个提案中，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，他将研究这一过程启动的数百个（如果不是数千个的话）位点是如何在适当的时间被选择和激活以开始染色体复制的。这些发现将提供与包括人类在内的所有动物细胞的增殖有关的重要信息。

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.