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Sequential ATP hydrolysis by Cdc6 and ORC directs loading of the Mcm2-7 helicase

John C W Randell1, Jayson L Bowers, Heather K Rodríguez

  • 1Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

Molecular Cell
|January 3, 2006
PubMed

Insights

Cdc6 ATP hydrolysis precedes ORC

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Eukaryotic DNA replication initiation requires Mcm2-7 helicase loading onto origins.
  • Prereplicative complex (pre-RC) formation involves ORC, Cdc6, Cdt1, and ATP.
  • ORC ATP hydrolysis is crucial for multiple Mcm2-7 loading rounds.

Purpose of the Study:

  • Investigate the role of Cdc6 ATP hydrolysis in pre-RC assembly.
  • Determine the precise step at which Cdc6 functions during pre-RC formation.

Main Methods:

  • Biochemical assays to study ATPase activity of Cdc6.
  • Inhibition of Cdc6 ATP hydrolysis to observe effects on pre-RC components.
  • Analysis of Mcm2-7 loading and Cdt1 stability.

Main Results:

  • Cdc6 acts as an ORC- and origin DNA-dependent ATPase.
  • Cdc6 hydrolysis occurs before ORC ATP hydrolysis.
  • Inhibiting Cdc6 ATP hydrolysis stabilizes Cdt1 and blocks Mcm2-7 loading.

Conclusions:

  • Cdc6 ATP hydrolysis is essential for a specific step in pre-RC assembly.
  • Distinct roles of ORC and Cdc6 ATP hydrolysis ensure regulated pre-RC formation.
  • This coordination is vital for temporal and spatial control of DNA replication initiation.

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