Cell cycle-dependent dynamic association of cyclin/Cdk complexes with human DNA replication proteins.
We have previously described the isolation of a replication competent (RC) complex from calf thymus, containing DNA polymerase alpha, DNA polymerase delta and replication factor C. Here, we describe the isolation of the RC complex from nuclear extracts of synchronized HeLa cells, which contains DNA replication proteins associated with cell-cycle regulation factors like cyclin A, cyclin B1, Cdk2 and Cdk1. In addition, it contains a kinase activity and DNA polymerase activities able to switch from a distributive to a processive mode of DNA synthesis, which is dependent on proliferating cell nuclear antigen. In vivo cross-linking of proteins to DNA in synchronized HeLa cells demonstrates the association of this complex to chromatin. We show a dynamic association of cyclins/Cdks with the RC complex during the cell cycle. Indeed, cyclin A and Cdk2 associated with the complex in S phase, and cyclin B1 and Cdk1 were present exclusively in G(2)/M phase, suggesting that the activity, as well the localization, of the RC complex might be regulated by specific cyclin/Cdk complexes.
Frouin
Isabelle
I
Istituto di Genetica Molecolare-CNR, Pavia, Italy.
Montecucco
Alessandra
A
Biamonti
Giuseppe
G
Hübscher
Ulrich
U
Spadari
Silvio
S
Maga
Giovanni
G
D000818:Animals; D002417:Cattle; D002453:Cell Cycle / Q000502:physiology*; D002467:Cell Nucleus / Q000502:physiology / Q000648:ultrastructure; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D004256:DNA Polymerase I / Q000378:metabolism*; D004258:DNA Polymerase III / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051818:Replication Protein C
D016213:Cyclins; D004268:DNA-Binding Proteins; D018844:Cyclin-Dependent Kinases; D004256:DNA Polymerase I; D004258:DNA Polymerase III; D051818:Replication Protein C