A Xenopus Dbf4 homolog is required for Cdc7 chromatin binding and DNA replication.
BACKGROUND
Early in the cell cycle a pre-replicative complex (pre-RC) is assembled at each replication origin. This process involves the sequential assembly of the Origin Recognition Complex (ORC), Cdc6, Cdt1 and the MiniChromosome Maintenance (Mcm2-7) proteins onto chromatin to license the origin for use in the subsequent S phase. Licensed origins must then be activated by S phase-inducing cyclin-dependent kinases (S-CDKs) and the Dbf4/Cdc7 kinase.


RESULTS
We have cloned a Xenopus homologue of Dbf4 (XDbf4), the sequence of which confirms the results of Furukhori et al. We have analysed the role of XDbf4 in DNA replication using cell-free extracts of Xenopus eggs. Our results indicate that XDbf4 is the regulatory subunit of XCdc7 required for DNA replication. We show that XDbf4 binds to chromatin during interphase, but unlike XCdc7, its chromatin association is independent of pre-RC formation, occurring in the absence of licensing, XCdc6 and XORC. Moreover, we show that the binding of XCdc7 to chromatin is dependent on the presence of XDbf4, whilst under certain circumstances XDbf4 can bind to chromatin in the absence of XCdc7. We provide evidence that the chromatin binding of XDbf4 that occurs in the absence of licensing depends on checkpoint activation.


CONCLUSIONS
We have identified XDbf4 as a functional activator of XCdc7, and show that it is required to recruit XCdc7 to chromatin. Our results also suggest that XCdc7 and XDbf4 are differentially regulated, potentially responding to different cell cycle signals.
Jares
Pedro
P
Wellcome Trust Biocentre, University of Dundee, DD1 5EH, UK. pjares@clinic.ub.es
Luciani
M Gloria
MG
Blow
J Julian
JJ
D000595:Amino Acid Sequence; D000818:Animals; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D004261:DNA Replication / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014981:Xenopus; D029867:Xenopus Proteins / Q000737:chemistry / Q000502:physiology
D018797:Cell Cycle Proteins; D002843:Chromatin; C485678:DBF4 protein, human; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D029867:Xenopus Proteins; C489405:CDC7 protein, Xenopus; C068619:CDC7 protein, human; C489403:Cdc7 protein, mouse; D017346:Protein Serine-Threonine Kinases