Structural basis of Mcm2-7 replicative helicase loading by ORC-Cdc6 and Cdt1

Nat Struct Mol Biol. 2017 Mar;24(3):316-324. doi: 10.1038/nsmb.3372. Epub 2017 Feb 13.

Abstract

To initiate DNA replication, the origin recognition complex (ORC) and Cdc6 load an Mcm2-7 double hexamer onto DNA. Without ATP hydrolysis, ORC-Cdc6 recruits one Cdt1-bound Mcm2-7 hexamer, thus forming an ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) helicase-loading intermediate. Here we report a 3.9-Å structure of Saccharomyces cerevisiae OCCM on DNA. Flexible Mcm2-7 winged-helix domains (WHDs) engage ORC-Cdc6. A three-domain Cdt1 configuration embraces Mcm2, Mcm4, and Mcm6, thus comprising nearly half of the hexamer. The Cdt1 C-terminal domain extends to the Mcm6 WHD, which binds the Orc4 WHD. DNA passes through the ORC-Cdc6 and Mcm2-7 rings. Origin DNA interaction is mediated by an α-helix within Orc4 and positively charged loops within Orc2 and Cdc6. The Mcm2-7 C-tier AAA+ ring is topologically closed by an Mcm5 loop that embraces Mcm2, but the N-tier-ring Mcm2-Mcm5 interface remains open. This structure suggests a loading mechanism of the first Cdt1-bound Mcm2-7 hexamer by ORC-Cdc6.

Publication types

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

MeSH terms

  • Binding Sites
  • Cell Cycle Proteins / chemistry*
  • Cell Cycle Proteins / metabolism
  • Cell Cycle Proteins / ultrastructure
  • Cryoelectron Microscopy
  • DNA Replication*
  • DNA, Fungal / chemistry
  • DNA, Fungal / metabolism
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / metabolism
  • DNA-Binding Proteins / ultrastructure
  • Mass Spectrometry
  • Minichromosome Maintenance Proteins / chemistry*
  • Minichromosome Maintenance Proteins / metabolism
  • Minichromosome Maintenance Proteins / ultrastructure
  • Models, Molecular
  • Nucleotides / metabolism
  • Protein Binding
  • Protein Domains
  • Protein Multimerization
  • Protein Structure, Secondary
  • Replication Origin*
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / metabolism
  • Saccharomyces cerevisiae Proteins / ultrastructure

Substances

  • CDC6 protein, S cerevisiae
  • Cell Cycle Proteins
  • DNA, Fungal
  • DNA-Binding Proteins
  • Nucleotides
  • Saccharomyces cerevisiae Proteins
  • TAH11 protein, S cerevisiae
  • Minichromosome Maintenance Proteins