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Originally published In Press as doi:10.1074/jbc.M207226200 on August 15, 2002

J. Biol. Chem., Vol. 277, Issue 43, 40520-40527, October 25, 2002
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Protein Phosphatase 2A Regulates Binding of Cdc45 to the Prereplication Complex*

Danny M. ChouDagger , Paris PetersenDagger , Johannes C. Walter§, and Gernot WalterDagger

From the Dagger  Department of Pathology, University of California at San Diego, La Jolla, California 92093 and the § Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

In eukaryotic cells, an ordered sequence of events leads to the initiation of DNA replication. During the G1 phase of the cell cycle, a prereplication complex (pre-RC) consisting of ORC, Cdc6, Cdt1, and MCM2-7 is established at replication origins on the chromatin. At the G1/S transition, MCM10 and the protein kinases Cdc7-Dbf4 and Cdk2-cyclin E cooperate to recruit Cdc45 to the pre-RC, followed by origin unwinding, RPA binding, and recruitment of DNA polymerases. Using the soluble DNA replication system derived from Xenopus eggs, we demonstrate that immunodepletion of protein phosphatase 2A (PP2A) from egg extracts and inhibition of PP2A activity by okadaic acid abolish loading of Cdc45 to the pre-RC. Consistent with a defect in Cdc45 loading, origin unwinding and the loading of RPA and DNA polymerase alpha  are also inhibited. Inhibition of PP2A has no effect on MCM10 loading and on Cdc7-Dbf4 or Cdk2 activity. The substrate of PP2A is neither a component of the pre-RC nor Cdc45. Instead, our data suggest that PP2A functions by dephosphorylating and activating a soluble factor that is required to recruit Cdc45 to the pre-RC. Furthermore, PP2A appears to counteract an unknown inhibitory kinase that phosphorylates and inactivates the same factor. Thus, the initiation of eukaryotic DNA replication is regulated at the level of Cdc45 loading by a combination of stimulatory and inhibitory phosphorylation events.

* This work was supported by United States Public Health Service Grant CA-36111.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pathology 0612, University of California at San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0612. Tel.: 858-534-1894; Fax: 858-534-8942; E-mail: gwalter@ucsd.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.


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