Abstract
Minichromosome maintenance (Mcm) proteins perform essential functions regulating the replication of chromatin. Human cells, like other eukaryotic cells, express at least six Mcm proteins conserved in the central region. We have earlier described the primary structures of five human Mcm proteins, but the primary structure of the sixth human Mcm protein, MCM6, was identified only recently. We now use antibodies, specific for the MCM6 protein, to assess its intranuclear distribution. We find that a fraction of MCM6 protein occurs in the nucleosol, forming multiprotein complexes with other Mcm proteins. More importantly, we use for the first time micrococcal nuclease as a tool to investigate the association of MCM6 protein with chromatin. After short digestion times, a considerable fraction of the MCM6 protein is released from chromatin as a multiprotein complex that includes other Mcm proteins as well. In addition, fractions of MCM3 and MCM6 proteins are released by nuclease digestion as monomeric proteins indicating that at least these two Mcm proteins may also occur as single molecules on chromatin. The data also suggest that the chromatin regions with bound Mcm proteins are more vulnerable to nuclease attack than bulk chromatin and may therefore differ in the arrangement of nucleosomes.
Highlights
We conclude that MCM6 protein (MCM6p) resides in chromatin regions that differ in structure or organization from bulk chromatin
Since the antigenic peptide corresponds to a section of MCM6p that is not conserved among Minichromosome maintenance (Mcm) proteins, we expected that the antibodies obtained are directed against MCM6p while ignoring the other Mcm proteins
The MCM3p-specific antibodies reacted with a polypeptide band (Fig. 1B) that migrated slightly slower under the electrophoretic conditions used and could be clearly distinguished from MCM6p
Summary
Thommes et al [15] detected the first mammalian Mcm protein by serendipity, whereas Hu et al [16] used an antibody directed against a central protein region, common to all Mcm proteins, to isolate additional Mcm sequences from a human HeLa cell cDNA library. 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 conclude that MCM6p resides in chromatin regions that differ in structure or organization from bulk chromatin
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