title: Isolation of the Cdc45/Mcm2-7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase.
abstract: 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.
authors.affiliation: Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720.
authors.forename: Stephen E
authors.initials: SE
authors.lastname: Moyer
authors.affiliation: Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720.
authors.forename: Peter W
authors.initials: PW
authors.lastname: Lewis
authors.affiliation: Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720 mbotchan@berkeley.edu.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Botchan
mesh_terms: 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
chemical_list: D018797:Cell Cycle Proteins; D029721:Drosophila Proteins; D010455:Peptides; D004247:DNA; C099190:CASK protein, Drosophila; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004265:DNA Helicases