Association of cyclin A and cdk2 with SV40 DNA in replication initiation complexes is cell cycle dependent |
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Authors: | Dominique Cannella James M Roberts Rati Fotedar |
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Institution: | (1) Institut de Biologie Structurale J.-P. Ebel, 41 Avenue des Martyrs, F-38027 Grenoble, Cedex 1, France, FR;(2) Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98104, USA, US |
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Abstract: | The cell cycle is driven by the sequential activation of a family of cyclin-dependent kinases (CDK) in association with cyclins.
In mammalian cells the timing of activation of cyclin A-associated kinase activity coincides with the onset of DNA synthesis
in S-phase. Using in vitro replication of SV40 origin-containing DNA as a model system, we have analyzed the proteins associated
with DNA during initiation of DNA replication in S-phase cell extracts. This analysis reveals that, in addition to replication
initiation proteins, cyclin A and cdk2 are also specifically associated with DNA. The association of cyclin A and cdk2 with
DNA during initiation is cell cycle regulated and occurs specifically in the presence of SV40 origin-containing plasmid and
SV40 T antigen (the viral replication initiator protein). The interactions among proteins involved in initiation play an important
role in DNA replication. We therefore investigated the ability of cyclin A and cdk2 to associate with replication initiation
proteins. Under replication initiation conditions, cyclin A and cdk2 from S-phase extracts specifically associate with SV40
T antigen. Further, the interaction of cyclin A-cdk2 with SV40 T antigen is mediated via cyclin A, and purified recombinant
cyclin A associates directly with SV40 T antigen. Taken together, our results suggest that cyclin A and cdk2 are components
of the SV40 replication initiation complex, and that protein-protein interactions between cyclin A-cdk2 and T antigen may
facilitate the association of cyclin A-cdk2 with the complex.
Received: 30 July 1996; in revised form: 25 September 1996 / Accepted: 8 October 1996 |
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