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.
