Regulation of MCM helicase loading and activation in the model system Saccharomyc

在每个细胞周期的S期确保基因组的完整和忠实复制是细胞分裂的关键。了解染色体DNA复制的过程，以及协调DNA复制与细胞周期的机制，将为诊断，预后和治疗水平的广泛临床研究工作提供基础。这个建议的长期目标是了解MCM解旋酶功能的真核细胞染色体DNA复制的模式系统酿酒酵母的调节。本申请提出了以下具体目标，以实现长期目标。 具体目标1：确定MCM双六聚体复合物形成对体内DNA复制的重要性。使用分子和遗传技术，MCM双六聚体复合物形成的DNA复制的重要性将被测试。这一目标的结果将提供重要的信息MCM解旋酶功能的机械机制，这是细胞周期调控的主要目标控制真核DNA复制。此外，这一目标将能够直接解决相互冲突的数据，并区分解旋酶功能的多个拟议模型。 具体目标2：确定DDK活性对DNA复制的主要功能作用是否是将Cdc 45加载到原始染色质上。MCM 5的突变体mcm 5-bob 1（P83 L）导致在早期G1中的组成性Cdc 45染色质加载和绕过G1-S转换和DNA复制起始所需的Dbf 4依赖性激酶（DDK）。该目的将确定Cdc 45染色质加载是否是DDK活性的最终下游顶点并且足以实现DDK旁路。对DNA复制控制的深入了解将为疾病状态提供更多的见解，其中DNA复制控制机制的失败导致染色体不稳定，突变和非整倍体，所有这些都是人类疾病的标志，包括出生缺陷，过早衰老和癌症。

Ensuring the complete and faithful duplication of the genome during S-phase of each cell cycle is key for cellular division. Understanding the process of chromosomal DNA replication, and the mechanisms that coordinate DNA replication with the cell cycle, will provide the basis for a wide range of clinical research efforts at the diagnostic, prognostic, and therapeutic levels. The long-term objective of this proposal is to understand the regulation of MCM helicase function for eukaryotic chromosomal DNA replication using the model system Saccharomyces cerevisiae. This application proposes the following specific aims to achieve the long-term objective. Specific Aim 1: To determine the importance of MCM double hexamer complex formation for in vivo DNA replication. Using both molecular and genetic techniques, the importance of MCM double hexamer complex formation for DNA replication will be tested. The results of this aim will provide important information on the mechanical mechanism of MCM helicase function, which is a primary target of cell cycle regulation controlling eukaryotic DNA replication. Furthermore, this aim will be able to directly address conflicting data and differentiate between multiple proposed models for helicase function. Specific Aim 2: To determine if the primary functional role of DDK activity for DNA replication is to load Cdc45 onto origin chromatin. A mutant of MCM5, mcm5-bob1 (P83L), results in constitutive Cdc45 chromatin loading in early G1 and bypass of required Dbf4 dependent kinase (DDK) for G1-S transition and initiation of DNA replication. This aim will determine if Cdc45 chromatin loading is the final downstream culmination of DDK activity and sufficient to achieve DDK bypass. An enhanced understanding of DNA replication control will provide additional insight into disease states where the failure of DNA replication control mechanisms leads to chromosomal instability, mutations, and aneuploidy, all of which are hallmarks of human disease including birth defects, premature ageing, and cancer.