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Volume 271, Number 49, Issue of December 6, 1996 pp. 31627-31637
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Role for Cyclin A-dependent Kinase in DNA Replication in Human S Phase Cell Extracts

(Received for publication, May 28, 1996, and in revised form, August 27, 1996)

Arun Fotedar , Dominique Cannella ^¶ , Patrick Fitzgerald , Tristan Rousselle ^¶ , Sunita Gupta ^¶ , Marcel Dorée and Rati Fotedar ^¶

From the  Division of Molecular Biology, La Jolla Institute of Allergy and Immunology, La Jolla, California 92037, USA the ^¶ Institut de Biologie Structurale, J.-P. Ebel, 41 Avenue des Martyrs, F-38027 Grenoble, Cedex 1, France and the  CNRS UPR 9008, Centre de Recherches de Biochimie Macromoleculaire, B.P. 5051, 1919 route de Mende 34033 Montpellier Cedex, France

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.

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