Association of cyclin A and cdk2 with SV40 DNA in replication initiation complexes is cell cycle dependent.
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.
Cannella
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Institut de Biologie Structurale J.-P. Ebel, 41 Avenue des Martyrs, F-38027 Grenoble, Cedex 1, France.
Roberts
J M
JM
Fotedar
R
R
D000818:Animals; D000957:Antigens, Viral, Tumor / Q000235:genetics / Q000378:metabolism; D016203:CDC2 Protein Kinase / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D042846:CDC2-CDC28 Kinases; D002453:Cell Cycle / Q000235:genetics*; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism*; D016213:Cyclins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D004257:DNA Polymerase II / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D016193:G1 Phase / Q000235:genetics; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D018741:Replication Origin; D051716:Replication Protein A; D016196:S Phase / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
D000957:Antigens, Viral, Tumor; D016213:Cyclins; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D051716:Replication Protein A; D014764:Viral Proteins; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; D042846:CDC2-CDC28 Kinases; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D004257:DNA Polymerase II