Colocalization of MCM8 and MCM7 with proteins involved in distinct aspects of DNA replication.
Minichromosome maintenance (MCM) proteins are essential for DNA replication in eukaryotes. A subcomplex of the MCM2-7 family members, initially characterized in yeast, is thought to serve as a eukaryotic DNA replicative helicase. MCM8 is a new family member, not present in yeast, which may function alone or with other family members in aspects of DNA metabolism, including replication initiation and elongation. Through the use of chromatin immunoprecipitation, we find that MCM8, like MCM7, colocalizes on a specific DNA segment of the c-MYC replication initiation zone (c-MYC replicator) with Cdc6, a protein potentially involved in loading MCM proteins onto DNA. The association between MCM8 and MCM7 peaks in mid G1, at the time of assembly of the prereplication complex. The association of both MCM proteins with Cdc6, however, continues even after DNA replication is complete. We also find that MCM8 colocalizes at the c-MYC replicator with chromatin-bound Cdk2. Our data indicate that any role MCM8 may play in elongation is likely to be discontinuous, in its association with DNA, from a potential role in initiation. Using immunogold electron microscopy we show that MCM8 and MCM7 differ in spatial relation to RPA70 during S phase. Our data strongly suggest that MCM8 functions with other known replication proteins in processes which accompany DNA replication, especially initiation, and which are specifically adapted to suit higher eukaryotes.
Kinoshita
Yayoi
Y
Department of Pathology, Mount Sinai School of Medicine, New York, New York 10029, USA.
Johnson
Edward M
EM
Gordon
Ronald E
RE
Negri-Bell
Heather
H
Evans
Mark T
MT
Coolbaugh
Jennifer
J
Rosario-Peralta
Yaseris
Y
Samet
Joseph
J
Slusser
Evelyn
E
Birkenbach
Mark P
MP
Daniel
Dianne C
DC
D018797:Cell Cycle Proteins / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D064168:Minichromosome Maintenance Complex Component 7; D064110:Minichromosome Maintenance Proteins; D009687:Nuclear Proteins / Q000378:metabolism*; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D051716:Replication Protein A / Q000378:metabolism
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C489427:MYC protein, human; D009687:Nuclear Proteins; D016271:Proto-Oncogene Proteins c-myc; C497167:RPA1 protein, human; D051716:Replication Protein A; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; C101624:MCM7 protein, human; C471623:MCM8 protein, human; D064168:Minichromosome Maintenance Complex Component 7; D064110:Minichromosome Maintenance Proteins