INITIATION OF DNA REPLICATION BY PHOSPHORYLATION

描述（来自申请人摘要的逐字逐句）： 这个实验室的目的是阐明控制 真核生物中的DNA复制在这个启动过程中有两个关键步骤 （1）复制前复合物（pre-Rcs）组装到 染色质;和（2）前RC的活化。ORC、Cdc 6和Mcm蛋白 是参与前RC形成的起始因子。这 该提案涉及磷酸化在组装和功能中的作用， 前RC。特别地，两种preRc组分Cdc 6 Mcm 4蛋白将被仔细研究。使用非洲爪蟾无细胞 系统中，Cdc 6蛋白的细胞周期磷酸化将被抑制。 其特征在于，其对蛋白质功能的影响通过产生一组 磷酸化突变体（特异性目的1）。Cdc 6可能介导 细胞周期蛋白依赖性激酶（Cdks）对前Rc组装的负面作用将是 测试.在特异性目标2中，Mcm 4蛋白内的磷酸化位点 将被映射，识别相关激酶和Mcm 4的作用 磷酸化对Mcm复合物功能的影响。最后在 特异性目的3 Cdc 7激酶在DNA起始过程中的作用 将探讨复制问题。Cdc 7是激酶的可能性 负责Mcm 4蛋白的磷酸化，同时它与 将检测S期的染色质。对前RCS的监管可能会 是细胞增殖和细胞分裂的最终承诺点之一。 控制的目标，确保交替的S-和M-阶段和 控制DNA复制检查点机制。因此， 拟议的实验将揭示细胞分裂的过程，因为它 在发育过程中和健康动物中正常发生， 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.