Role for cyclin A-dependent kinase in DNA replication in human S phase cell extracts.
Cell cycle progression is regulated by cyclin-dependent kinases. Using in vitro replication of SV40 origin containing DNA as a model system, we have performed a detailed analysis of the dependence on cyclin-associated kinases of mammalian DNA replication. Complete immunodepletion of cyclin A from human S phase cell extracts decreases replication, and replication activity of cyclin A-depleted S phase extracts can subsequently be restored by the addition of purified CDK2-cyclin A kinase. Addition of cyclin A alone reconstitutes both kinase activity and DNA replication, whereas addition of cyclin E or cyclin B reconstitutes neither. We therefore conclude that reconstitution of DNA replication specifically correlates with an increase in kinase activity. By comparison, depletion of cyclin E from S phase cell extracts does not have any significant inhibitory effect on DNA replication. Moreover, specific p21(Waf1) mutants that bind to CDK2-cyclin and inhibit both cyclin A and cyclin E kinase activities, but do not bind to proliferating cell nuclear antigen, inhibit DNA replication to the same extent as cyclin A depletion. Together, these results show that the kinase activity associated with cyclin A, but not with cyclin E, is primarily responsible for activating SV40 plasmid replication in mammalian S phase cell extracts. Finally, we present evidence that the cyclin-dependent kinase does not influence the assembly of initiation complexes but acts at a stage prior to elongation.
Fotedar
A
A
Division of Molecular Biology, La Jolla Institute of Allergy and Immunology, La Jolla, California 92037, USA.
Cannella
D
D
Fitzgerald
P
P
Rousselle
T
T
Gupta
S
S
Dorée
M
M
Fotedar
R
R
D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D016213:Cyclins / Q000378:metabolism; D004261:DNA Replication; D006801:Humans; D010441:Peptide Chain Elongation, Translational; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D016196:S Phase; D014407:Tumor Cells, Cultured
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D018809:Proliferating Cell Nuclear Antigen; D011494:Protein Kinases; C102333:histone H1 kinase; D018844:Cyclin-Dependent Kinases