Initiation of DNA Replication of Yeast Chromosomes

描述(申请人提供)：基因组的及时和准确复制对于所有真核细胞的正常增殖是必不可少的。因此，DNA复制的启动与细胞周期的进展密切相关。这项提议的长期目标是确定选择DNA复制地点并启动启动过程的事件。DNA复制的控制与复制前复合体(Pre-RC)的形成和激活密切相关，复制前复合体包括起源识别复合体(ORC)、CDC6和McM2-7复合体(可能的复制型DNA解旋酶)。使用新的体外前RC形成试验和ORC、CDC6和McM2-7蛋白突变体的组合，贝尔博士将：1.确定前RC的组成和前RC形成的机制。2.确定ATP如何控制前RC的形成。3.确定细胞周期蛋白依赖的激酶和ORC如何协同工作，以防止起源在细胞周期中的不适当时间重新启动。4.开发新的体外检测方法，用于检测从特定复制起点引发复制的事件。这些研究将使用酿酒酵母进行，然而，真核DNA复制的高度保守性表明，酵母的进展将迅速转化为对人类细胞中相同过程的理解。在酿酒酵母中取得的快速进展使其成为进行这些研究的理想生物。对这些蛋白质的新理解将导致抗真菌化合物的强大候选靶点和识别癌细胞的潜在标记物。此外，在这些研究中获得的对酵母复制蛋白的了解将指导研究确定这些蛋白的人类类似物的抑制剂，这些蛋白代表着新的化疗化合物的重要靶点。

DESCRIPTION (provided by applicant): The timely and accurate replication of the genome is essential to the normal proliferation of all eukaryotic cells. Accordingly the initiation of DNA replication is carefully coordinated with the progress of the cell cycle. The long-term objective of this proposal is to determine the events that select the sites of DNA replication and activate the initiation process. Control of DNA replicaiton is tightly linked to the formation and activation of the pre-replicative complex (pre-RC) that includes the origin recognition complex (ORC), Cdc6 and the Mcm2-7 complex (the putative replicative DNA helicase). Using a combination of new in vitro assays for pre- RC formation and mutants in ORC, Cdc6 and the Mcm2-7 proteins Dr. Bell will: 1. Determine the components of the pre-RC and the mechanism of pre-RC formation. 2. Determine the how ATP controls the formation of the pre-RC. 3. Determine how cyclin dependent kinases and ORC work together to prevent origins from re-initiating at inappropriate times during the cell cycle. 4. Develop new in vitro assays for the events leading to replication initiation from a defined origin of replication. These studies will be performed using the yeast S. cerevisiae, however, the highly conserved nature of eukaryotic DNA replication indicates progress in yeast will rapidly translated to an understanding of the same processes in human cells. The rapid progress that can be made in S. cerevisiae make it an ideal organism to perform these studies. New understanding of these proteins will lead to strong candidate targets for antifungal compounds and potential markers to identify cancer cells. In addition, the understanding of the yeast replication proteins gained in these studies will direct studies to identify inhibitors of the human analogs of these proteins, which represent important targets for novel chemotherapeutic compounds.