Structural mechanism of helicase loading onto replication origin DNA by ORC-Cdc6

Proc Natl Acad Sci U S A. 2020 Jul 28;117(30):17747-17756. doi: 10.1073/pnas.2006231117. Epub 2020 Jul 15.


DNA replication origins serve as sites of replicative helicase loading. In all eukaryotes, the six-subunit origin recognition complex (Orc1-6; ORC) recognizes the replication origin. During late M-phase of the cell-cycle, Cdc6 binds to ORC and the ORC-Cdc6 complex loads in a multistep reaction and, with the help of Cdt1, the core Mcm2-7 helicase onto DNA. A key intermediate is the ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) complex in which DNA has been already inserted into the central channel of Mcm2-7. Until now, it has been unclear how the origin DNA is guided by ORC-Cdc6 and inserted into the Mcm2-7 hexamer. Here, we truncated the C-terminal winged-helix-domain (WHD) of Mcm6 to slow down the loading reaction, thereby capturing two loading intermediates prior to DNA insertion in budding yeast. In "semi-attached OCCM," the Mcm3 and Mcm7 WHDs latch onto ORC-Cdc6 while the main body of the Mcm2-7 hexamer is not connected. In "pre-insertion OCCM," the main body of Mcm2-7 docks onto ORC-Cdc6, and the origin DNA is bent and positioned adjacent to the open DNA entry gate, poised for insertion, at the Mcm2-Mcm5 interface. We used molecular simulations to reveal the dynamic transition from preloading conformers to the loaded conformers in which the loading of Mcm2-7 on DNA is complete and the DNA entry gate is fully closed. Our work provides multiple molecular insights into a key event of eukaryotic DNA replication.

Keywords: ATPase; DNA replication; Mcm2-7 helicase; cryo-EM; origin recognition complex.

Publication types

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

MeSH terms

  • Binding Sites
  • Cell Cycle Proteins / chemistry*
  • Cell Cycle Proteins / metabolism*
  • Cryoelectron Microscopy
  • DNA Helicases / chemistry*
  • DNA Helicases / metabolism*
  • DNA Replication*
  • Minichromosome Maintenance Complex Component 6 / chemistry
  • Minichromosome Maintenance Complex Component 6 / metabolism
  • Molecular Docking Simulation
  • Molecular Dynamics Simulation
  • Origin Recognition Complex
  • Protein Binding
  • Protein Conformation
  • Replication Origin*
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Structure-Activity Relationship


  • CDC6 protein, S cerevisiae
  • Cell Cycle Proteins
  • Origin Recognition Complex
  • Saccharomyces cerevisiae Proteins
  • DNA Helicases
  • MCM6 protein, S cerevisiae
  • Minichromosome Maintenance Complex Component 6