J. Biochem, 2003, Vol. 134, No. 3 447-457
© 2003 Japanese Biochemical Society
MOLECULAR BIOLOGY |
Identification and Characterization of a Xenopus Homolog of Dbf4, a Regulatory Subunit of the Cdc7 Protein Kinase Required for the Initiation of DNA Replication
1 Research Institute for Microbial Diseases, 2 Graduate School of Science, 3 Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871; 4 Institute of Medical Science, University of Tokyo, Tokyo 108-8639; and 5 Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613
Dbf4 is a regulatory subunit for the Cdc7 protein kinase that is required for the initiation of eukaryotic DNA replication, but the precise roles of Dbf4-Cdc7 remain to be determined. Here we identified a Xenopus homolog of Dbf4 (XDbf4) and characterized XDbf4 and Xenopus Cdc7 (XCdc7) in Xenopus egg extracts. XDbf4 formed a complex with XCdc7 in egg extracts and activated XCdc7 kinase activity in vitro. In contrast with Dbf4 in yeast and mammalian cultured cells, the XDbf4 levels in egg extracts did not change during the cell cycle progression. XDbf4 was a phosphoprotein in interphase extracts, and was apparently hyperphosphorylated in cytostatic factor (CSF)-mediated, metaphase-arrested extracts and in mitotic extracts. However, the hyperphosphorylation of XDbf4 did not seem to affect the level of kinase activation, or chromatin binding of the XDbf4-XCdc7 complex. Upon release from CSF-arrest, XDbf4 was partially dephosphorylated and bound to chromatin. Interestingly, XDbf4 was loaded onto chromatin before XCdc7 during DNA replication in egg extracts. These results suggest that the function of XDbf4-XCdc7 during the early embryonic cell cycle is regulated in a manner distinct from that during the somatic cell cycle.
* To whom correspondence should be addressed. Chromosome Replication Group, Laboratories for Biomolecular Network, Graduate School of Frontier Biosciences, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871. Tel: +81-6-6879-8332, Fax: +81-6-6877-3584, E-mail: swaga{at}biken.osaka-u.ac.jp
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