Isolation of the Cdc45/Mcm2-7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase.
The protein Cdc45 plays a critical but poorly understood role in the initiation and elongation stages of eukaryotic DNA replication. To study Cdc45's function in DNA replication, we purified Cdc45 protein from Drosophila embryo extracts by a combination of traditional and immunoaffinity chromatography steps and found that the protein exists in a stable, high-molecular-weight complex with the Mcm2-7 hexamer and the GINS tetramer. The purified Cdc45/Mcm2-7/GINS complex is associated with an active ATP-dependent DNA helicase function. RNA interference knock-down experiments targeting the GINS and Cdc45 components establish that the proteins are required for the S phase transition in Drosophila cells. The data suggest that this complex forms the core helicase machinery for eukaryotic DNA replication.
Moyer
Stephen E
SE
Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720.
Lewis
Peter W
PW
Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720.
Botchan
Michael R
MR
Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720 mbotchan@berkeley.edu.
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002846:Chromatography, Affinity; D004247:DNA / Q000096:biosynthesis; D004265:DNA Helicases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004261:DNA Replication; D029721:Drosophila Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D005057:Eukaryotic Cells / Q000201:enzymology; D010455:Peptides / Q000494:pharmacology; D011485:Protein Binding; D016196:S Phase
D018797:Cell Cycle Proteins; D029721:Drosophila Proteins; D010455:Peptides; D004247:DNA; C099190:CASK protein, Drosophila; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004265:DNA Helicases