Stepwise Regulated Chromatin Assembly of MCM2–7 Proteins*
- From the Institute of Human Genetics, CNRS, Genome Dynamics and Development, 141 rue de la Cardonille, Montpellier, Cedex 5, France
Abstract
Acquisition of the competence to replicate requires the assembly of the MCM2–7 (minichromosome maintenance) protein complex onto pre-replicative chromatin, a step of the licensing reaction. This step is thought to occur through binding of a heterohexameric MCM complex containing the six related MCM subunits. Here we show that assembly of the MCM complex onto pre-replicative chromatin occurs through sequential stabilization of specific MCM subunits. Inhibition of licensing with 6-dimethylaminopurine results in chromatin containing specifically bound MCM4 and MCM6. A similar result was obtained by interference of the assembly reaction with an MCM3 antibody. The presence of chromatin-bound MCM intermediates was confirmed by reconstitution experiments in vitro with purified proteins and by the observation of an ordered association of MCM subunits with chromatin. These results indicate that the assembly of the MCM complex onto pre-replicative chromatin is regulated at the level of distinct subunits, suggesting an additional regulatory step in the formation of pre-replication complexes.
Footnotes
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↵* This work was supported in part by grants from the Association pour la Recherche sur le Cancer and the Fondation de la Recherche Médicale.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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↵‡ Supported by fellowships from the European Community (Biomedicine and Health) and the Fondation pour la Recherche Médicale.
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↵§ To whom correspondence should be addressed. Tel.: 33-0-499619917; Fax: 33-0-499619920; E-mail: mechali@igh.cnrs.fr.
- Abbreviations:
- ORC
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origin recognition complex
- MCM
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minichromosome maintenance
- CDC
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cell division cycle
- 6-DMAP
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6-dimethylaminopurine
- LSS
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low speed supernatant
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- Received November 23, 1999.
- Revision received January 12, 2000.
- The American Society for Biochemistry and Molecular Biology, Inc.
