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 c yclin-dependent kinases (CDK) in association with cyclins. In mammalia n cells the timing of activation of cyclin A-associated kinase activit y 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 rep lication in S-phase cell extracts. This analysis reveals that, in addi tion to replication initiation proteins, cyclin A and cdk2 are also sp ecifically associated with DNA. The association of cyclin A and cdk2 w ith DNA during initiation is cell cycle regulated and occurs specifica lly in the presence of SV40 origin-containing plasmid and SV40 T antig en (the viral replication initiator protein). The interactions among p roteins involved in initiation play an important role in DNA replicati on. We therefore investigated the ability of cyclin A and cdk2 to asso ciate with replication initiation proteins. Under replication initiati on conditions, cyclin A and cdk2 from S-phase extracts specifically as sociate with SV40 T antigen. Further, the interaction of cyclin A-cdk2 with SV40 T antigen is mediated via cyclin A, and purified recombinan t cyclin A associates directly with SV40 T antigen. Taken together, ou r results suggest that cyclin A and cdk2 are components of the SV40 re plication initiation complex, and that protein-protein interactions be tween cyclin A-cdk2 and T antigen may facilitate the association of cy clin A-cdk2 with the complex.