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Molecular and Cellular Biology, February 1999, p. 1226-1241, Vol. 19, No. 2
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Cyclin B-Cdk1 Kinase Stimulates ORC- and Cdc6-Independent Steps of Semiconservative Plasmid Replication in Yeast Nuclear Extracts

Bernard P. Duncker,1 Philippe Pasero,1,dagger Diego Braguglia,1,Dagger Patrick Heun,1 Michael Weinreich,2 and Susan M. Gasser1,*

Swiss Institute for Experimental Cancer Research, CH-1066 Epalinges, Switzerland,1 and Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 117242

Received 7 August 1998/Returned for modification 14 September 1998/Accepted 26 October 1998

Nuclear extracts from Saccharomyces cerevisiae cells synchronized in S phase support the semiconservative replication of supercoiled plasmids in vitro. We examined the dependence of this reaction on the prereplicative complex that assembles at yeast origins and on S-phase kinases that trigger initiation in vivo. We found that replication in nuclear extracts initiates independently of the origin recognition complex (ORC), Cdc6p, and an autonomously replicating sequence (ARS) consensus. Nonetheless, quantitative density gradient analysis showed that S- and M-phase nuclear extracts consistently promote semiconservative DNA replication more efficiently than G1-phase extracts. The observed semiconservative replication is compromised in S-phase nuclear extracts deficient for the Cdk1 kinase (Cdc28p) but not in extracts deficient for the Cdc7p kinase. In a cdc4-1 G1-phase extract, which accumulates high levels of the specific Clb-Cdk1 inhibitor p40SIC1, very low levels of semiconservative DNA replication were detected. Recombinant Clb5-Cdc28 restores replication in a cdc28-4 S-phase extract yet fails to do so in the cdc4-1 G1-phase extract. In contrast, the addition of recombinant Xenopus CycB-Cdc2, which is not sensitive to inhibition by p40SIC1, restores efficient replication to both extracts. Our results suggest that in addition to its well-characterized role in regulating the origin-specific prereplication complex, the Clb-Cdk1 complex modulates the efficiency of the replication machinery itself.

* Corresponding author. Mailing address: Swiss Institute for Experimental Cancer Research, Ch. des Boveresses 155, CH-1066 Epalinges, Switzerland. Phone: 41 21 692 5886. Fax: 41 21 652 6933. E-mail: sgasser{at}eliot.unil.ch.

dagger Present address: IGM UMR 5535, CNRS, 34033 Montpellier, France.

Dagger Present address: SulzerInnotec Ltd., CH-8401 Winterthur, Switzerland.

Molecular and Cellular Biology, February 1999, p. 1226-1241, Vol. 19, No. 2
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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