BM28, a human member of the MCM2-3-5 family, is displaced from chromatin during DNA replication.
We have recently cloned and characterized a human member (BM28) of the MCM2-3-5 family of putative relication factors (Todorov, I.T., R. Pepperkok, R.N. Philipova, S. Kearsey, W. Ansorge, and D. Werner. 1994. J. Cell Sci. 107:253-265). While this protein is located in the nucleus throughout interphase, we report here a dramatic alteration in its nuclear binding during the cell cycle. BM28 is retained in the nucleus after Triton X-100 extraction in G1 and early S phase cells, but is progressively lost as S phase proceeds, and little BM28 is retained in detergent-extracted G2 nuclei. BM28 that is resistant to extraction in G1 nuclei is removed by DNase I digestion, suggesting that the protein is chromatin associated. In addition, we present evidence for variations in the electrophoretic mobility of BM28 that may reflect posttranslational modifications of BM28 during the cell cycle. During mitosis, BM28 is present as a fast-migrating form, but on entry into G1, the protein is converted into a slow-migrating form. With the onset of S phase, the slow-migrating form is progressively converted into the fast form. BM28 is phosphorylated at all stages of the cell cycle, but during interphase the fast form is hyperphosphorylated compared with the slow form. These apparent changes in modification may reflect or effect changes in the nuclear binding of BM28. The behavior of BM28 is not dissimilar to related proteins in Saccharomyces cerevisiae, such as Mcm2p, which are excluded from the nucleus after DNA replication. We speculate that BM28 may be involved in the control that limits eukaryotic DNA replication to one round per cell cycle.
Todorov
I T
IT
Department of Zoology, University of Oxford, United Kingdom.
Attaran
A
A
Kearsey
S E
SE
D016475:3T3 Cells; D000818:Animals; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D002843:Chromatin / Q000378:metabolism* / Q000648:ultrastructure; D004261:DNA Replication; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D064111:Minichromosome Maintenance Complex Component 2; D008961:Models, Structural; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding
D018797:Cell Cycle Proteins; D002843:Chromatin; D005656:Fungal Proteins; D009687:Nuclear Proteins; C084859:MCM2 protein, human; D064111:Minichromosome Maintenance Complex Component 2