Initiation of DNA Replication of Yeast Chromosomes

描述（由申请人提供）：基因组的及时和准确复制对所有真核细胞的正常增殖至关重要。因此，DNA复制的开始与细胞周期的进展密切协调。这项建议的长期目标是确定选择DNA复制位点和激活起始过程的事件。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（假定的复制DNA解旋酶）。使用ORC， Cdc6和Mcm2-7蛋白的预RC形成和突变的新体外检测组合，Bell博士将：1。确定预rc的组成和预rc的形成机制。2. 确定ATP如何控制pre-RC的形成。3. 确定细胞周期蛋白依赖性激酶和ORC如何协同工作，以防止在细胞周期中不适当的时间重新启动起源。4. 开发新的体外实验，从一个确定的复制起点开始导致复制。这些研究将使用酵母酿酒酵母进行，然而，真核生物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相同过程的理解。酿酒葡萄球菌的快速进展使其成为进行这些研究的理想生物。对这些蛋白质的新认识将为抗真菌化合物和潜在标志物识别癌细胞提供强有力的候选靶点。此外，在这些研究中获得的对酵母复制蛋白的理解将指导研究确定这些蛋白的人类类似物的抑制剂，这些类似物代表了新型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.