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PMID: 9891057 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Cyclin B-cdk1 kinase stimulates ORC- and Cdc6-independent steps of semiconservative plasmid replication in yeast nuclear extracts.

Molecular and cellular biology ·Vol. 19 ·No. 2 ·1999-02-00 ·Pages 1226-41

Duncker BP, Pasero P, Braguglia D, Heun P, Weinreich M, Gasser SM

Abstract

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 p40(SIC1), 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 p40(SIC1), 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.

MeSH Terms
Animals CDC2 Protein Kinase/genetics,metabolism Cell Cycle Proteins/genetics,metabolism Cell Nucleus/metabolism Cyclin B/genetics,metabolism DNA Replication/genetics DNA, Fungal/biosynthesis,genetics DNA-Binding Proteins/genetics,metabolism Fungal Proteins/genetics,metabolism G1 Phase In Vitro Techniques Mutation Origin Recognition Complex Plasmids/genetics,metabolism Recombinant Proteins/genetics,metabolism S Phase Saccharomyces cerevisiae/genetics,metabolism Saccharomyces cerevisiae Proteins Xenopus
Chemicals
CDC6 protein, S cerevisiae Cell Cycle Proteins Cyclin B DNA, Fungal DNA-Binding Proteins Fungal Proteins Origin Recognition Complex Recombinant Proteins Saccharomyces cerevisiae Proteins CDC2 Protein Kinase
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Duncker B P
Swiss Institute for Experimental Cancer Research, CH-1066 Epalinges, Switzerland.
Pasero P
Braguglia D
Heun P
Weinreich M
Gasser S M
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.19.2.1226
Published
1999-02-00
Pages
1226-41
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC116052
Subset
IM
Grants
NIGMS NIH HHS · GM-45436 · United States
External Links
PubMed source DOI: 10.1128/MCB.19.2.1226 PMC Full Text View journal details
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