Wei Theng Poh1,2,†, Gaganmeet Singh Chadha1,†, Peter J. Gillespie1, Philipp Kaldis2 and J. Julian Blow1
1 Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dow St., Dundee DD1 5EH, UK
2 Institute of Molecular and Cell Biology, Agency for Science, Technology, and Research (A*STAR), Singapore 138673, Republic of Singapore
† These authors contributed equally to this study.
Published in Open Biology on 8 January 2014.
The initiation of DNA replication requires two protein kinases: cyclin-dependent kinase (Cdk) and Cdc7. Although S phase Cdk activity has been intensively studied, relatively little is known about how Cdc7 regulates progression through S phase. We have used a Cdc7 inhibitor, PHA-767491, to dissect the role of Cdc7 in Xenopus egg extracts. We show that hyperphosphorylation of mini-chromosome maintenance (MCM) proteins by Cdc7 is required for the initiation, but not for the elongation, of replication forks. Unlike Cdks, we demonstrate that Cdc7 executes its essential functions by phosphorylating MCM proteins at virtually all replica- tion origins early in S phase and is not limiting for progression through the Xenopus replication timing programme. We demonstrate that protein phos- phatase 1 (PP1) is recruited to chromatin and rapidly reverses Cdc7-mediated MCM hyperphosphorylation. Checkpoint kinases induced by DNA damage or replication inhibition promote the association of PP1 with chromatin and increase the rate of MCM dephosphorylation, thereby counteracting the previously com- pleted Cdc7 functions and inhibiting replication initiation. This novel mechanism for regulating Cdc7 function provides an explanation for previous contradictory results concerning the control of Cdc7 by checkpoint kinases and has implications for the use of Cdc7 inhibitors as anti-cancer agents.
Figure Legend: Schematic diagram of the role and regulation of Cdc7 in Xenopus egg extracts. (a) A single replication origin is shown which is licensed by loading a double hexamer of Mcm2 – 7 proteins (‘MCM’). This promotes ELYS binding and the recruitment of Cdc7 to Mcm2 – 7 at the origin. (b) Events occurring during S phase are shown at both an early-replicating (left) and a late-replicating origin (right). Mcm2–7 at both origins are phosphorylated by Cdc7, which in turn is reversed by PP1. Cdk substrates for initiation are recruited preferentially to the early-replicating origin. Checkpoint kinases activated in response to etoposide or other inhibitors promote PP1 chromatin association with reverse Cdc7 activity but may also independently inhibit Cdk activity.
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