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

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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.