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An ORC/Cdc6/MCM2-7 complex is formed in a multistep reaction to serve as a platform for MCM double-hexamer assembly

Alejandra Fernández-Cid1, Alberto Riera, Silvia Tognetti

  • 1DNA Replication Group, MRC Clinical Sciences Centre, Imperial College, London W12 0NN, UK.

Molecular Cell
|April 23, 2013
PubMed

Insights

DNA replication requires loading the MCM2-7 helicase. This study reveals how Cdt1 and ATP hydrolysis by Orc1/Cdc6 overcome Mcm6 inhibition, forming the ORC/Cdc6/MCM2-7 complex for efficient helicase loading.

Area of Science:

  • Cellular biology
  • Molecular biology
  • Biochemistry

Background:

  • DNA replication initiation is crucial for cell division.
  • The MCM2-7 helicase loading involves ORC, Cdc6, and Cdt1.
  • The precise mechanism of MCM2-7 double-hexamer formation remains unclear.

Purpose of the Study:

  • To elucidate the mechanism of MCM2-7 helicase loading onto DNA.
  • To identify the roles of Mcm6 autoinhibition and Cdt1 in helicase loading.
  • To investigate the function of Orc1 and Cdc6 ATP hydrolysis in complex formation.

Main Methods:

  • Biochemical assays to study protein interactions and activities.
  • Analysis of Mcm6 autoinhibition and Cdt1's role in overcoming it.
  • Investigating the impact of Orc1/Cdc6 ATP hydrolysis on complex assembly.

Main Results:

  • Mcm6's C-terminal domain autoinhibitor blocks MCM2-7 recruitment by ORC/Cdc6.
  • Cdt1 overcomes Mcm6 inhibition, activating ORC/Cdc6 ATP hydrolysis for helicase loading.
  • Orc1 ATP hydrolysis, alongside Cdc6, is critical for ORC/Cdc6/MCM2-7 (OCM) complex formation and MCM2-7 double-hexamer assembly.
  • CDK-dependent ORC phosphorylation inhibits OCM formation, ensuring cell-cycle-specific replication.

Conclusions:

  • This study reveals novel mechanisms regulating DNA replication licensing.
  • Cdt1 and Orc1/Cdc6 ATP hydrolysis are key regulators of MCM2-7 loading.
  • The findings provide a redefined understanding of the DNA licensing process.

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