Incorporation into the prereplicative complex activates the Mcm2–7 helicase for Cdc7–Dbf4 phosphorylation
- Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
Abstract
The essential S-phase kinase Cdc7–Dbf4 acts at eukaryotic origins of replication to trigger a cascade of protein associations that activate the Mcm2–7 replicative helicase. Also known as Dbf4-dependent kinase (DDK), this kinase preferentially targets chromatin-associated Mcm2–7 complexes that are assembled on the DNA during prereplicative complex (pre-RC) formation. Here we address the mechanisms that control the specificity of DDK action. We show that incorporation of Mcm2–7 into the pre-RC increased the level and changes the specificity of DDK phosphorylation of this complex. In the context of the pre-RC, DDK preferentially targets a conformationally distinct subpopulation of Mcm2–7 complexes that is tightly linked to the origin DNA. This targeting requires DDK to tightly associate with Mcm2–7 complexes in a Dbf4-dependent manner. Importantly, we find that DDK association with and phosphorylation of origin-linked Mcm2–7 complexes require prior phosphorylation of the pre-RC. Our findings provide insights into the mechanisms that ensure that DDK action is spatially and temporally restricted to the origin-bound Mcm2–7 complexes that will drive replication fork movement during S phase and suggest new mechanisms to regulate origin activity.
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Footnotes
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↵1 Present address: University of Puerto Rico, Mayaguez, Puerto Rico 00681-9000, USA.
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↵2 Corresponding author.
↵E-MAIL spbell{at}mit.edu; FAX (617) 253-4043.
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Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1759609.
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Supplemental material is available at http://www.genesdev.org.
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- Received November 4, 2008.
- Accepted January 21, 2009.
- Copyright © 2009 by Cold Spring Harbor Laboratory Press