In vivo interaction of human MCM heterohexameric complexes with chromatin. Possible involvement of ATP.
The MCM protein family, which consists of at least six members, has been implicated in the regulatory machinery causing DNA to replicate once in the S phase. Mammalian MCM proteins are present in the nucleus in two different forms, one extractable by nonionic detergents and the other resistant to such extraction. The latter is assumed to be tightly associated with nuclear structures and released at the time of initiation of replication. However, details of the mode of binding remain unclear. In the present study, we found that, in nonionic detergent-permeabilized nuclei, the association of human MCM (hMCM) proteins with them could be stabilized by the addition of ATP. The hMCMs bound to the nuclei in the presence of ATP were released by digestion with nucleases, suggesting that they are chromatin-associated. The nuclease-directed solubilization of the chromatin-bound hMCMs thus provided a means to analyze them as well as soluble hMCMs by co-immunoprecipitation. The results indicate that the six hMCM members exist as heterocomplexes, whether bound or unbound. We therefore propose that hMCM proteins may function in DNA replication as heterohexamers associated with chromatin and that ATP is possibly involved in the association. Nuclease digestion-immunoprecipitation techniques of the type described here should facilitate further elucidation of the mode of interaction between hMCMs and chromatin.
Fujita
M
M
Laboratory of Viral Oncology, Research Institute, Aichi Cancer Center, Chikusa-ku, Nagoya 464, Japan. mfujita@aichigw.aichi-cc.pref.aichi.jp
Kiyono
T
T
Hayashi
Y
Y
Ishibashi
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000906:Antibodies; D018797:Cell Cycle Proteins / Q000302:isolation & purification / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism* / Q000648:ultrastructure; D004261:DNA Replication; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D046911:Macromolecular Substances; D015636:Magnesium Chloride / Q000494:pharmacology; D064168:Minichromosome Maintenance Complex Component 7; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism; D012265:Ribonucleotides / Q000494:pharmacology; D013816:Thermodynamics
D000906:Antibodies; D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D012265:Ribonucleotides; D015636:Magnesium Chloride; D000255:Adenosine Triphosphate; C101624:MCM7 protein, human; D064168:Minichromosome Maintenance Complex Component 7