Mobilization of chromatin-bound Mcm proteins by micrococcal nuclease.

Mcm (minichromosome maintenance) proteins are important components of the eukaryotic replication initiation apparatus. We investigate the binding of human Mcm proteins to HeLa cell chromatin using micrococcal nuclease as a tool. In previous work we prepared chromatin under low ionic strength conditions. The use of a low salt buffer was necessary to prevent the dissociation of Mcm proteins. Here we use chromatin prepared at more physiological salt concentrations (100 mM NaCl) following the procedure of Fujita et al. (J. Biol. Chem. 272, 10928-10935; 1997) who had shown that ATP stabilizes the interaction of Mcm proteins with chromatin. We show here that micrococcal nuclease released Mcm proteins early during the digestion process suggesting that Mcm proteins reside on chromatin sites which are more open to nuclease attack than bulk chromatin. Released Mcm proteins sedimented through glycerol gradients as a multiprotein complex comprising several of the six known human Mcm proteins.

Stability of the replicative Mcm3 protein in proliferating and differentiating human cells.

Mcm proteins are abundant nuclear proteins involved in the regulation of genome replication. Previous experiments had shown that levels of Mcm-specific mRNAs increase at the G1/S phase transition of the cell cycle, but that the amounts of Mcm proteins do not change much during the cell cycle. To learn more about the stability of an Mcm protein we performed experiments which showed that: (i) more than 60% of [35S]methionine pulse-labeled Mcm3 protein appears to be degraded during a 24-h chase in HeLa cells; (ii) the amount of Mcm3 protein significantly decreases during the differentiation of HL60 cells in vitro (whereas another replication-initiation protein, hOrc2, remains fairly constant); and (iii) according to immunohistochemical staining, Mcm3 protein is present in nuclei of cells in the proliferating zone of human epidermal tissue, but in decreasing amounts in nuclei of differentiating cells of the upper cell layers. Our interpretation is that Mcm3 protein is no longer synthesized after initiation of differentiation and slowly disappears at a half-life of approximately 24 h.

Human protein MCM6 on HeLa cell chromatin.

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.

A novel human Mcm protein: homology to the yeast replication protein Mis5 and chromosomal location.

Mcm proteins perform functions related to the regulation of eukaryotic genome replication. Previous work has shown that human cells contain at least five different Mcm proteins. We report now the amino acid sequence of an additional human Mcm protein, p105Mcm, and show that it is homologous to the Schizosaccharomyces pombe protein Mis5. We demonstrate that the gene for protein p105Mcm (HGMW-approved symbol, MCM6) is located on human chromosome 2q14-q21. All six known human Mcm proteins have now been sequenced and compared to their yeast counterparts.