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

在每个细胞周期的S阶段，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。这项建议的长期目标是利用酿酒酵母模型系统来了解MCM解旋酶功能对真核细胞染色体DNA复制的调节。这项申请提出了以下具体目标，以实现长期目标。 具体目的1：确定MCM双六聚体复合体的形成对体内DNA复制的重要性。利用分子和遗传技术，将测试MCM双六聚体复合体形成对DNA复制的重要性。这一目的的结果将为MCM解旋酶功能的机械机制提供重要的信息，MCM解旋酶功能是控制真核DNA复制的细胞周期调控的主要靶点。此外，这一目标将能够直接处理相互冲突的数据，并区分解旋酶功能的多个拟议模型。 具体目的2：确定DDK活性对DNA复制的主要功能作用是否是将CDC45装载到起始染色质上。MCM5的突变体MCM5-Bob1(P83L)导致在G1期早期结构性地装载CDC45染色质，并绕过了G1-S转换所需的Dbf4依赖激酶。这一目标将决定CDC45染色质负载是否是DDK活性的最终下游顶峰，并足以实现DDK旁路。对DNA复制控制的进一步了解将提供对疾病状态的进一步洞察，在这些疾病状态下，DNA复制控制机制的失败会导致染色体不稳定、突变和非整倍体，所有这些都是人类疾病的特征，包括出生缺陷、早衰和癌症。

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.