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Originally published In Press as doi:10.1074/jbc.M108118200 on November 26, 2001

J. Biol. Chem., Vol. 277, Issue 4, 2702-2708, January 25, 2002
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A Potential Role for Mini-chromosome Maintenance (MCM) Proteins in Initiation at the Dihydrofolate Reductase Replication Origin*

Mark G. AlexandrowDagger , Marion Ritzi§, Alexander PemovDagger , and Joyce L. HamlinDagger

From the Dagger  Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia 22908 and the § Department of Biochemistry, University of Konstanz, Postfach 5560, D-78434, Konstanz, Germany

Mini-chromosome maintenance (MCM) proteins were originally identified in yeast, and homologues have been identified in several other eukaryotic organisms, including mammals. These findings suggest that the mechanisms by which eukaryotic cells initiate and regulate DNA replication have been conserved throughout evolution. However, it is clear that many mammalian origins are much more complex than those of yeast. An example is the Chinese hamster dihydrofolate reductase (DHFR) origin, which resides in the spacer between the DHFR and 2BE2121 genes. This origin consists of a broad zone of potential sites scattered throughout the 55-kb spacer, with several subregions (e.g. ori-beta , ori-beta ', and ori-gamma ) being preferred. We show here that antibodies to human MCMs 2-7 recognize counterparts in extracts prepared from hamster cells; furthermore, co-immunoprecipitation data demonstrate the presence of an MCM2-3-5 subcomplex as observed in other species. To determine whether MCM proteins play a role in initiation and/or elongation in Chinese hamster cells, we have examined in vivo protein-DNA interactions between the MCMs and chromatin in the DHFR locus using a chromatin immunoprecipitation (ChIP) approach. In synchronized cultures, MCM complexes associate preferentially with DNA in the intergenic initiation zone early in S-phase during the time that replication initiates. However, significant amounts of MCMs were also detected over the two genes, in agreement with recent observations that the MCM complex co-purifies with RNA polymerase II. As cells progress through S-phase, the MCMs redistribute throughout the DHFR domain, suggesting a dynamic interaction with DNA. In asynchronous cultures, in which replication forks should be found at any position in the genome, MCM proteins were distributed relatively evenly throughout the DHFR locus. Altogether, these data are consistent with studies in yeast showing that MCM subunits localize to origins during initiation and then migrate outward with the replication forks. This constitutes the first evidence that mammalian MCM complexes perform a critical role during the initiation and elongation phases of replication at the DHFR origin in hamster cells.

* This work was supported by National Institutes of Health Grant R01 GM 26108 (to J. L. H.) and National Institutes of Health postdoctoral fellowship 5F32 GM19304-02 (to M. G. A.).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.

To whom correspondence should be addressed: University of Virginia School of Medicine, Box 800733 HSC, Charlottesville, VA 22908. Tel.: 804-924-5858; E-mail: jlh2d@virginia.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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