Premature Structural Changes at Replication Origins in a Yeast Minichromosome Maintenance (MCM) Mutant*
- From the ‡Departments of Microbiology and Molecular Genetics and ‖Pathology and the §Markey Center for Molecular Genetics, University of Vermont School of Medicine, Burlington, Vermont 05405
Abstract
The Cdc7p protein kinase in the budding yeastSaccharomyces cerevisiae is thought to help trigger DNA replication by modifying one or more of the factors that assemble at replication origins (ARSs). To investigate events catalyzed by Cdc7p, we compared the structure of replication origins in cells containing conditional mutations in Cdc7p and Cdc8p, a thymidylate kinase that is required for DNA synthesis. High resolution genomic footprinting indicated that the presumptive lagging strand template in ARS1 became highly sensitive to KMnO4 modification after theCDC7 execution point. These results suggested that Cdc7p triggers DNA unwinding. The transition from late G1 phase to the CDC7 execution point and from the CDC7to the CDC8 execution points was accompanied by small but ARS-dependent changes in DNA topology. These results suggested that DNA unwinding before the CDC8 execution point either is highly localized or that the torsional stress associated with initial DNA unwinding is minimized by compensatory protein-DNA structural changes. The ARS DNA structural attributes evident in cells blocked at the CDC8 execution point were also evident in α-factor-blocked, G1 phase cells containing the CDC7 bypass mutantmcm5/cdc46-bob1. This result strongly suggests that the structural changes during the transition from the CDC7 toCDC8 execution points depend on the Cdc7p protein kinase and involve alteration of the minichromosome maintenance protein complex.
Footnotes
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↵* This work was supported by a grant from the Lake Champlain Cancer Research Organization and National Institutes of Health Grant GM52017 (to D. S. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵¶ Present address: Rogers and Wells, 200 Park Ave., New York, NY, 10166.
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↵** To whom correspondence should be addressed: Dept. of Microbiology and Molecular Genetics, University of Vermont School of Medicine, Stafford Hall, Rm. 302, Burlington, Vermont 05405. Tel.: 802-656-8586; Fax: 802-656-8749; E-mail: dpederso@zoo.uvm.edu.
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Published, JBC Papers in Press, April 4, 2000, DOI 10.1074/jbc.M909787199
- Abbreviations:
- ARS
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autonomously replicating sequences
- ACS
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ARS consensus sequence
- ORC
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origin recognition complex
- HU
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hydroxyurea
- MNase
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micrococcal nuclease
- bp
-
base pair(s)
- MCM
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minichromosome maintenance
- RPA
-
replication protein A
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- Received December 6, 1999.
- Revision received March 20, 2000.
- The American Society for Biochemistry and Molecular Biology, Inc.
