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Cell cycle-dependent phosphorylation of the DNA polymerase epsilon subunit, Dpb2, by the Cdc28 cyclin-dependent protein kinase

Academic Article
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Overview

authors

  • Kesti, T.
  • McDonald, W. H.
  • Yates III, John
  • Wittenberg, Curt

publication date

  • April 2004

journal

  • Journal of Biological Chemistry  Journal

abstract

  • DNA polymerase epsilon (Polepsilon), 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. Polepsilon is recruited to replication origins during late G(1) 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 G(1) 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 G(1) 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 G(1) phase at CDK consensus sites facilitates the interaction with Pol2 or the activity of Polepsilon

subject areas

  • CDC28 Protein Kinase, S cerevisiae
  • Cell Cycle
  • DNA Polymerase II
  • DNA Replication
  • Mutation
  • Phenotype
  • Phosphorylation
  • Saccharomycetales
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Identity

International Standard Serial Number (ISSN)

  • 0021-9258

Digital Object Identifier (DOI)

  • 10.1074/jbc.M313289200

PubMed ID

  • 14747467
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Additional Document Info

start page

  • 14245

end page

  • 14255

volume

  • 279

issue

  • 14

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