Isolation of the Cdc45/Mcm2-7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase

Proc Natl Acad Sci U S A. 2006 Jul 5;103(27):10236-10241. doi: 10.1073/pnas.0602400103. Epub 2006 Jun 23.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Calcium-Calmodulin-Dependent Protein Kinases / genetics
  • Calcium-Calmodulin-Dependent Protein Kinases / isolation & purification*
  • Calcium-Calmodulin-Dependent Protein Kinases / metabolism*
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / isolation & purification*
  • Cell Cycle Proteins / metabolism*
  • Chromatography, Affinity
  • DNA / biosynthesis
  • DNA Helicases / genetics
  • DNA Helicases / isolation & purification*
  • DNA Helicases / metabolism*
  • DNA Replication*
  • Drosophila Proteins / genetics
  • Drosophila Proteins / isolation & purification*
  • Drosophila Proteins / metabolism*
  • Drosophila melanogaster / enzymology
  • Drosophila melanogaster / genetics
  • Drosophila melanogaster / metabolism
  • Eukaryotic Cells / enzymology
  • Peptides / pharmacology
  • Protein Binding
  • S Phase

Substances

  • Cell Cycle Proteins
  • Drosophila Proteins
  • Peptides
  • DNA
  • CASK protein, Drosophila
  • Calcium-Calmodulin-Dependent Protein Kinases
  • DNA Helicases