Human Protein MCM6 on HeLa Cell Chromatin

Abstract

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...

Dynamic Localization of the DNA Replication Proteins MCM5 and MCM7 in Plants

“…5E). Similar results have been reported in yeast, mouse, and frog, where MCM4, MCM6, and MCM7 form a "core" complex that is more tightly associated with chromatin than the remaining subunits (Ishimi et al, 1996;Coue et al, 1998;Holthoff et al, 1998;Lee and Hurwitz, 2000;Prokhorova and Blow, 2000;Davey et al, 2003).…”

“…In parallel assays, the majority of the chromatin marker HMGA remained associated with the pellet fraction, establishing that our extraction protocol does not result in widespread dissociation of chromatin and cannot account for the bulk of MCM5 and MCM7 in the soluble fraction. In asynchronous, immortalized human cells, the nonchromatin-bound fraction of MCM ranges from slightly more than half (Holthoff et al, 1998;Mendez and Stillman, 2000) to approximately 90% of the total cellular protein (Burkhart et al, 1995). A significant fraction of the non-chromatin-bound MCM in the Arabidopsis culture may have originated from cells that have exited the cell cycle.…”

Identification of two residues in MCM5 critical for the assembly of MCM complexes and Stat1-mediated transcription activation in response to IFN-γ

“…Earlier studies using immunoprecipitation, gradient centrifugation, and gel filtration techniques have shown that the MCM proteins form various subcomplexes composed of different members of the family, such as MCM5͞3 or MCM4͞6͞7 subcomplexes at varying salt concentrations (40,41,(43)(44)(45)(46). To see the pattern of MCM complexes and Stat1 in IFN-␥-treated cells, nuclear extracts were prepared from BUD-8 cells untreated or treated with IFN-␥ for 30 min and subjected to gel filtration followed by SDS͞PAGE and Western blotting analyses.…”

“…The MCMs form complexes of various constitution (35,40,44), assemble at the initiation site of DNA replication along with other protein complexes such as originrecognition complex (48), and travel together with the replisome (49,50). The MCMs have common structural features such as DNA-dependent ATPase domains characteristic of DNA helicases (39).…”

The DNA replication factor MCM5 is essential for Stat1-mediated transcriptional activation

“…The molecular structure and in vitro analyses of these proteins suggest that they function as a DNA helicase (3); they form a heterohexamer complex that binds to DNA replication origins and moves along with the DNA polymerase during DNA replication elongation (4,5). In addition to the hexamer complex, the MCM proteins also form subcomplexes containing some members of the family, such as MCM4͞6͞7 or MCM3͞5 (3,(6)(7)(8). It has been suggested that these subcomplexes represent segments during the assembly of the hexameric MCM complex (9).…”