title: Interactions of the human MCM-BP protein with MCM complex components and Dbf4.
abstract: MCM-BP was discovered as a protein that co-purified from human cells with MCM proteins 3 through 7; results which were recapitulated in frogs, yeast and plants. Evidence in all of these organisms supports an important role for MCM-BP in DNA replication, including contributions to MCM complex unloading. However the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood. Here we show that human MCM-BP is capable of interacting with individual MCM proteins 2 through 7 when co-expressed in insect cells and can greatly increase the recovery of some recombinant MCM proteins. Glycerol gradient sedimentation analysis indicated that MCM-BP interacts most strongly with MCM4 and MCM7. Similar gradient analyses of human cell lysates showed that only a small amount of MCM-BP overlapped with the migration of MCM complexes and that MCM complexes were disrupted by exogenous MCM-BP. In addition, large complexes containing MCM-BP and MCM proteins were detected at mid to late S phase, suggesting that the formation of specific MCM-BP complexes is cell cycle regulated. We also identified an interaction between MCM-BP and the Dbf4 regulatory component of the DDK kinase in both yeast 2-hybrid and insect cell co-expression assays, and this interaction was verified by co-immunoprecipitation of endogenous proteins from human cells. In vitro kinase assays showed that MCM-BP was not a substrate for DDK but could inhibit DDK phosphorylation of MCM4,6,7 within MCM4,6,7 or MCM2-7 complexes, with little effect on DDK phosphorylation of MCM2. Since DDK is known to activate DNA replication through phosphorylation of these MCM proteins, our results suggest that MCM-BP may affect DNA replication in part by regulating MCM phosphorylation by DDK.
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Canada.
authors.forename: Tin
authors.initials: T
authors.lastname: Nguyen
authors.forename: Madhav
authors.initials: M
authors.lastname: Jagannathan
authors.forename: Kathy
authors.initials: K
authors.lastname: Shire
authors.forename: Lori
authors.initials: L
authors.lastname: Frappier
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D003902:Detergents / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D064111:Minichromosome Maintenance Complex Component 2; D064148:Minichromosome Maintenance Complex Component 4; D064151:Minichromosome Maintenance Complex Component 6; D064168:Minichromosome Maintenance Complex Component 7; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016196:S Phase; D012492:Salts / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; C485678:DBF4 protein, human; D004268:DNA-Binding Proteins; D003902:Detergents; C519407:MCMBP protein, human; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D012492:Salts; C084859:MCM2 protein, human; C099243:MCM4 protein, human; C103258:MCM6 protein, human; C101624:MCM7 protein, human; D064111:Minichromosome Maintenance Complex Component 2; D064148:Minichromosome Maintenance Complex Component 4; D064151:Minichromosome Maintenance Complex Component 6; D064168:Minichromosome Maintenance Complex Component 7