Cryo-EM Structure of a Helicase Loading Intermediate Containing ORC-Cdc6-Cdt1-MCM2-7 Bound to DNA

Nat Struct Mol Biol. 2013 Aug;20(8):944-51. doi: 10.1038/nsmb.2629. Epub 2013 Jul 14.

Abstract

In eukaryotes, the Cdt1-bound replicative helicase core MCM2-7 is loaded onto DNA by the ORC-Cdc6 ATPase to form a prereplicative complex (pre-RC) with an MCM2-7 double hexamer encircling DNA. Using purified components in the presence of ATP-γS, we have captured in vitro an intermediate in pre-RC assembly that contains a complex between the ORC-Cdc6 and Cdt1-MCM2-7 heteroheptamers called the OCCM. Cryo-EM studies of this 14-subunit complex reveal that the two separate heptameric complexes are engaged extensively, with the ORC-Cdc6 N-terminal AAA+ domains latching onto the C-terminal AAA+ motor domains of the MCM2-7 hexamer. The conformation of ORC-Cdc6 undergoes a concerted change into a right-handed spiral with helical symmetry that is identical to that of the DNA double helix. The resulting ORC-Cdc6 helicase loader shows a notable structural similarity to the replication factor C clamp loader, suggesting a conserved mechanism of action.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Cycle Proteins / chemistry*
  • Cell Cycle Proteins / metabolism
  • Chromosomal Proteins, Non-Histone / chemistry*
  • Chromosomal Proteins, Non-Histone / metabolism
  • Cloning, Molecular
  • Cryoelectron Microscopy
  • DNA Replication / physiology
  • DNA, Fungal / chemistry
  • DNA, Fungal / metabolism
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / metabolism
  • Image Processing, Computer-Assisted
  • Immunoprecipitation
  • Models, Molecular*
  • Multiprotein Complexes / chemistry*
  • Mutagenesis, Site-Directed
  • Origin Recognition Complex / chemistry*
  • Origin Recognition Complex / metabolism
  • Protein Conformation*
  • Saccharomyces cerevisiae / chemistry*
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / metabolism

Substances

  • CDC6 protein, S cerevisiae
  • Cell Cycle Proteins
  • Chromosomal Proteins, Non-Histone
  • DNA, Fungal
  • DNA-Binding Proteins
  • Multiprotein Complexes
  • ORC1 protein, S cerevisiae
  • Origin Recognition Complex
  • Saccharomyces cerevisiae Proteins
  • TAH11 protein, S cerevisiae
  • MCM2 protein, S cerevisiae