Protein phosphatase 2A regulates binding of Cdc45 to the prereplication complex.
In eukaryotic cells, an ordered sequence of events leads to the initiation of DNA replication. During the G(1) 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 G(1)/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.
Department of Pathology 0612, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Danny M
DM
Chou
Paris
P
Petersen
Johannes C
JC
Walter
Gernot
G
Walter
D002352:Carrier Proteins / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D011485:Protein Binding; D054648:Protein Phosphatase 2; D018741:Replication Origin; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
C102209:CDC45 protein, S cerevisiae; D002352:Carrier Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2