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Mcm proteins have recently attracted considerable attention because they are thought to perform an essential function in the regulation of chromatin replication (reviewed in Refs. 1 and 2). Eukaryotic cells express six evolutionarily conserved Mcm proteins. They were so named because the first members of this family of nuclear proteins were discovered through genetic screens of yeast mutants unable to maintain the extrachromosomal replication of minichromosomes (Mcm, minichromosome maintenance) 1 (3). Mcm proteins are abundant nuclear proteins with copy numbers ranging 10 4 to 10 6 per cell nucleus. They occur either free in the nucleosol or are bound to nuclear chromatin. The fraction of chromatin-bound Mcm proteins is highest at the end of the G 1 phase or the beginning of the S phase of the cell cycle but gradually decreases as S phase proceeds (4 -6). In postreplicative cells, essentially all Mcm proteins are free in the nucleosol and disperse throughout the cell upon nuclear breakdown in mitosis, but they rapidly reassemble on chromatin sites in nuclei after mitotic telophase.Binding (19). A cDNA, encoding human MCM6p, was also isolated and described by Tsuruga et al. (20).Here, we describe the preparation of MCM6-specific antibodies which we have used to assess the distribution of MCM6p in the nucleus and the interaction of MCM6p with chromatin from HeLa cells. For this purpose, we degraded chromatin with micrococcal nuclease and separated the resulting chromatin fragments by glycerol gradient centrifugation. We partially recovered MCM6p in association with chromatin fragments while another part of MCM6p appeared as free protein. We conclude that MCM6p resides in chromatin regions that differ in structure or organization from bulk chromatin. EXPERIMENTAL PROCEDURESCell Growth and Cell Fractionation-HeLa cells were cultivated on plastic dishes unter previously described conditions (21). For synchronization, cells were arrested in early S phase by two subsequent thymidine blocks (22) and released into the cell cycle by removing excess thymidine. Progression through S phase was monitored by pulse-labeling with [ 3 H]-thymidine (1 Ci/60 min) (21). Incorporated radioactivity was determined in acid precipitates by scintillation counting. The number of mitotic cells was determined according to Ohyashiki et al. (23).Nuclei were prepared either in buffers with 40 g/ml digitonin essentially as described by Adam et al. (24), or after swelling of washed HeLa cells in hypotonic buffer A (10 mM Hepes, pH 7.5; 0.5 mM EDTA; 0.1% Nonidet P-40) by Dounce homogenization as described previously in detail (25). Protein extracts were prepared from digitonin nuclei by 0.5% Nonidet P-40 in 150 mM sodium acetate, 10 mM Tris-HCl (pH 7.5), and 1 mM EDTA. Unsoluble material was removed by centrifugation at 100,000 ϫ g. The supernatant was analyzed by immunoblotting (see below) or through glycerol gradient centrifugation (40 to 5% glycerol in the sodium acetate buffer described above).Chromatin Preparation-Chromatin was prepared as o...

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