Abstract 575: Cdk1 prevents DNA rereplication in G2/M by phosphorylating and facilitating the removal of Cdc7 from chromatin at the end of S phase.

Abstract Cancer is inextricably linked with aneuploidy. Aneuploidy can arise for a number of reasons, including the deregulation of DNA and centrosome replication. Mechanisms have evolved that are designed to ensure that DNA is replicated only once per cell cycle. The initiation of DNA replication is a key target of these mechanisms, which includes regulating Cdc6, Cdt1 and Cyclin dependent kinases (Cdks). Although the regulatory mechanisms of Cdc6 and Cdt1 are known, the role of Cdk1 in particular is less clear. One potential target for Cdk1 regulation is the Dbf4-Cdc7 kinase (DDK), which acts as a licensing factor for DNA replication. DDK performs an essential and critical step in initiating DNA replication. As such, it is a target for novel cancer therapeutic drugs currently in clinical trials. Even so, the regulatory mechanisms dictating DDK function in human cells remains poorly understood. A site-directed mutagenesis approach was undertaken to determine the in vivo function of Cdk-dependent phosphorylation sites on the primary Cdc7 sequence. Using over-expressed Cdc7 in human cells (HEK293T), the functional significance of Cdc7 phosphorylation was determined; both the lack of phosphorylation and constitutive phosphorylation of Cdc7 was analyzed. We found that Cdc7, the catalytic subunit of the DDK, is a target of Cdk1. Cdc7 is phosphorylated in G2/M phase by Cdk1. Phosphorylation of Cdc7 removes the catalytic subunit of DDK from origins of replication and chromatin with the assistance of the nuclear export factor CRM1, which relocates Cdc7 to the microtubule cytoskeleton. Finally, the Cdk1-dependent removal of Cdc7 from chromatin prevents the cell from initiating DNA replication, indicating that Cdc7 is a target of Cdk1-dependent inhibition of DNA re-replication. We conclude that (1) Cdk1 phosphorylates Cdc7 in vivo. (2) Cdk1 dependent phosphorylation of Cdc7 regulates chromatin binding and DNA replication and (3) pCdc7 is associated with the nuclear export factor CRM1 in G2/M phase, suggesting a mechanism that couples the down-regulation of Cdc7 function at the end of S phase through nuclear localization and chromatin association. Taken together, our data provides key insight into the functional understanding of DDK. Citation Format: James Knockleby, Byung Ju Kim, Hoyun Lee. Cdk1 prevents DNA rereplication in G2/M by phosphorylating and facilitating the removal of Cdc7 from chromatin at the end of S phase. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 575. doi:10.1158/1538-7445.AM2013-575