Characterization of the Yeast Cdc7p/Dbf4p Complex Purified from Insect Cells

doi:10.1074/jbc.M003491200

Characterization of the Yeast Cdc7p/Dbf4p Complex Purified from Insect Cells

ITS PROTEIN KINASE ACTIVITY IS REGULATED BY Rad53p*

From the ^‡Department of Biochemistry and Molecular Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan, the ^§Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247-0063, Japan, and the ^¶Division of Yeast Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom

Abstract

The yeast Saccharomyces cerevisiaeCdc7p/Dbf4p protein kinase complex was purified to near homogeneity from insect cells. The complex efficiently phosphorylated yeast Mcm2p and less efficiently the remaining Mcm proteins or other replication proteins. Significantly, when pretreated with alkaline phosphatase, Mcm2p became completely inactive as a substrate, suggesting that it must be phosphorylated by other protein kinase(s) to be a substrate for the Cdc7p/Dbf4p complex. Mutant Cdc7p/Dbf4p complexes containing either Cdc7-1p or Dbf4-1∼5p were also partially purified from insect cells and characterized in vitro. Furthermore, the autonomously replicating sequence binding activity of variousdbf4 mutants was also analyzed. These studies suggest that the autonomously replicating sequence-binding and Cdc7p protein kinase activation domains of Dbf4p collaborate to form an active Cdc7p/Dbf4p complex and function during S phase in S. cerevisiae. It is shown that Rad53p phosphorylates the Cdc7p/Dbf4p complex in vitro and that this phosphorylation greatly inhibits the kinase activity of Cdc7p/Dbf4p. This result suggests that Rad53p controls the initiation of chromosomal DNA replication by regulating the protein kinase activity associated with the Cdc7p/Dbf4p complex.

Footnotes

↵* This work was supported by a grant-in-aid for scientific research on priority area and for international collaboration research from the Ministry of Education, Science, Sport and Culture of Japan (to A. S.).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. Tel.: 81-6-6879-8331; Fax: 81-6-6877-3584; E-mail: asugino@biken.osaka-u.ac.jp.
Published, JBC Papers in Press, August 9, 2000, DOI 10.1074/jbc.M003491200
↵2 M. Kihara, W. Nakai, S. Asano, A. Suzuki, K. Kitada, Y. Kawasaki, L. H. Johnston, and A. Sugino, unpublished results.
↵4 M. Kihara and A. Sugino, unpublished results.
↵3 N. Nakashima, K. Hashimoto, and A. Sagino, umpublished results.
↵5 K. Kitada, M. Shinohara, and A. Sugino, unpublished results.
Abbreviations:

ARS

autonomously replicating sequence

HA

hemagglutinin

PAGE

polyacrylamide gel electrophoresis

GST

glutathione S-transferase

BAP

alkaline phosphatase

HU

hydroxyurea
- Received April 25, 2000.
- Revision received August 24, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.