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J. Biol. Chem., Vol. 279, Issue 14, 14245-14255, April 2, 2004

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Cell Cycle-dependent Phosphorylation of the DNA Polymerase Epsilon Subunit, Dpb2, by the Cdc28 Cyclin-dependent Protein Kinase^*

Tapio Kesti, W. Hayes McDonald, John R. Yates, III, and Curt Wittenberg¶

From the Departments of Molecular Biology and Cell Biology, The Scripps Research Institute, La Jolla, California 92037

DNA polymerase (Pol), one of the three major eukaryotic replicative polymerases, is comprised of the essential catalytic subunit, called Pol2 in budding yeast, and three accessory subunits, only one of which, Dpb2, is essential. Pol is recruited to replication origins during late G₁ phase prior to activation of replication. In this work we show that the budding yeast Dpb2 is phosphorylated in a cell cycle-dependent manner during late G₁ phase. Phosphorylation results in the appearance of a lower mobility species. The appearance of that species in vivo is dependent upon the Cdc28 cyclin-dependent protein kinase (CDK), which can directly phosphorylate Dpb2 in vitro. Either G₁ cyclin (Cln) or B-type cyclin (Clb)-associated CDK is sufficient for phosphorylation. Mapping of phosphorylation sites by mass spectrometry using a novel gel-based proteolysis protocol shows that, of the three consensus CDK phosphorylation sites, at least two, Ser-144 and Ser-616, are phosphorylated in vivo. The Cdc28 CDK phosphorylates only Ser-144 in vitro. Using site-directed mutagenesis, we show that Ser-144 is sufficient for the formation of the lower mobility form of Dpb2 in vivo. In contrast, Ser-616 appears not to be phosphorylated by Cdc28. Finally, inactivation of all three CDK consensus sites in Dpb2 results in a synthetic phenotype with the pol2-11 mutation, leading to decreased spore viability, slow growth, and increased thermosensitivity. We suggest that phosphorylation of Dpb2 during late G₁ phase at CDK consensus sites facilitates the interaction with Pol2 or the activity of Pol

Received for publication, December 5, 2003 , and in revised form, January 25, 2004.

^* This work was supported by National Science Foundation Grant MCB-00911211 (to C. W.) and American Cancer Society, California Division Fellowship Grant 1-13-00 (to T. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This 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 Molecular Biology, Scripps Research Inst., MB-3, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-9628; Fax: 858-784-2265.

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