Grant: Discovery Grants Program - Individual

My long-term research goal is unravelling the mechanism of how human cells maintain genetic stability in the context of DNA replication and cell cycle control. To maintain genetic stability, the entire genome must replicate faithfully and only once per cell cycle. The latter is achieved by strictly regulating the stepwise formation of the pre-replication complex (pre-RC; licensing) and the orderly activation of replication initiation. Available data indicate that Cdc7 plays critical roles for both licensing and replication initiation processes. Cdc7 itself is activated when it is bound by the Dbf4 regulatory subunit. However, the exact mechanism when Dbf4 binds to Cdc7 to activate it is poorly understood. A prevalent model has been that Cdc7 is activated by Cdk-mediated phosphorylation in the context of high levels of Dbf4. Contrarily to this model, however, we have recently found that non-phosphorylated Cdc7 has high affinity for the origin of DNA replication and activates DNA replication. Furthermore, Cdk1/cyclin B-mediated Cdc7 phosphorylation leads to its dissociation from chromatin, preventing DNA (re)replication and, thus, ensuring once-per-cell cycle DNA replication. We also found that Cdc7 as the replication activator is restored by PP1-mediated dephosphorylation as cells exit mitosis. PP1 has also been found to activate a replication checkpoint after pre-RC is already formed. Thus, PP1 may function as a replication promoter as well as an inhibitor: for the former by restoring Cdc7 function and the latter by activating replication checkpoint (i.e., inhibiting replication initiation). Thus, human cells need to have intimate crosstalk between Cdc7, Cdk1 and PP1 to maintain genetic stability. Based on these observations, we postulate that the PP1-mediated Cdc7 dephosphorylation is an active regulation mechanism to promote replication at two different steps: the licensing and initiation activation steps. To test this hypothesis, we propose to: (i) systematically examine the role of PP1-mediated Cdc7 dephosphorylation in the replication licensing and activation process in normal, immortalized, and cancerous human cells; and (ii) determine the potential role of Cdc7 dephosphorylation in the regulation of cytokinesis. Data from this study will shed new light on the mechanism of how human cells maintain or lose genetic stability in the context of DNA replication and cell cycle control.