INITIATION OF DNA REPLICATION BY PHOSPHORYLATION

描述(逐字摘自申请者摘要)：的长期目标 这个实验室是为了阐明控制启动的机制。 真核生物中的DNA复制。这一启动过程中的两个关键步骤 是：(1)将复制前复合体(Pre-RCs)组装到 染色质；以及(2)前RCS的激活。ORC、CDC6和MCM蛋白 是参与前RCS形成的启动因素。这 提案阐述了磷酸化在装配和功能中的作用 RCS前。特别是，两个前Rc组分--CDC6的磷酸化 并将对Mcm4蛋白进行密切研究。使用无细胞非洲爪哇 系统中，cdc6蛋白的细胞周期磷酸化将是 特征及其对生成集合所确定的蛋白质功能的影响 磷酸化突变体(特定目标1)。CDC6介导的可能性 细胞周期蛋白依赖性蛋白激酶(CDK)在前RC组装中的负面作用将是 测试过。在特定目标2中，Mcm4蛋白中的磷酸化位点 将作图，确定相关的激酶(S)和Mcm4的作用 对MCM复合体的功能进行了磷酸化测定。最后，在 特异靶3CDC7激酶在DNA起始过程中的作用 我们将探索复制。CDC7可能是一种蛋白激酶 负责Mcm4蛋白的磷酸化，而它结合到 对S生长期的染色质进行检测。对RCS前的监管可能会 是细胞增殖和物理的最终承诺点之一 确保S相和M相以及 DNA复制检查点机制的控制。因此， 拟议中的实验将阐明细胞分裂的过程，因为它 在发育期间和健康的动物中正常发生，而在 疾病过程，如癌症。

DESCRIPTION (Verbatim from the applicant's abstract): The long-term goal of this laboratory is to elucidate the mechanism that controls the initiation of DNA replication in eukaryotes. Two critical steps in this initiation process are: (1) the assembly of the pre-replication complexes (pre-Rcs) onto the chromatin; and (2) activation of the pre-Rcs. The ORC, Cdc6 and Mcm proteins are initiation factors that participate in the formation of the pre-Rcs. This proposal addresses the role of phosphorylation in the assembly and function of the pre-Rcs. In particular, phosphorylation of two preRc components, the Cdc6 and the Mcm4 proteins will be closely studied. Using a Xenopus cell-free system, the cell cycle phosphorylation of the Cdc6 protein will be characterized and its effect on protein function determined by generating a set of phosphorylation mutants (Specific Aim 1). The possibility that Cdc6 mediates the negative role of Cyclin-dependent kinases (Cdks) on pre-Rc assembly will be tested. In Specific Aim 2, the phosphorylation sites within the Mcm4 protein will be mapped, the relevant kinase(s) identified and the effect of Mcm4 phosphorylation on the function of the Mcm complex determined. Finally, in Specific Aim 3 the role of the Cdc7 kinase during the initiation of DNA replication will be explored. The possibility that Cdc7 is the kinase responsible for the phosphorylation of the Mcm4 protein while it is bound to chromatin during S-phase will be tested. Regulation of the pre-Rcs is likely to be one of the final commitment points for cell proliferation and the physical target of the controls that ensure alternation of the S- and M-phase and the controls for the DNA replication checkpoint machinery. Thus, the results of the proposed experiments will shed light on the process of cell division, as it occurs normally during development and in healthy animals, and abnormally in disease processes such as cancer.