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An Orc6 tether mediates ORC binding-site switching during replication origin licensing.

David Driscoll1,2, Larry J Friedman2,3, Jeff Gelles3

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The origin recognition complex (ORC) uses Orc6 to tether Mcm2-7 helicases during DNA replication initiation. This interaction ensures efficient loading of two helicases by one ORC, preventing its release and enabling cell division.

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • DNA replication initiation is a fundamental process for cell division.
  • The origin recognition complex (ORC) and Mcm2-7 helicases are crucial for loading replication machinery.
  • A single ORC is proposed to load two Mcm2-7 helicases at replication origins.

Purpose of the Study:

  • To elucidate the molecular mechanism of how one ORC loads two Mcm2-7 helicases.
  • To investigate the role of Orc6 in the helicase loading process.
  • To understand how CDK phosphorylation affects ORC function during origin licensing.

Main Methods:

  • Single-molecule Förster Resonance Energy Transfer (smFRET) assay.
  • Site-directed mutagenesis of Orc6.
  • Biochemical assays to monitor ORC-Mcm2-7 interactions.

Main Results:

  • The N-terminal region of Orc6 acts as a tether, connecting ORC to Mcm2 during the transition of ORC's binding site.
  • This tethering interaction is essential for preventing ORC release into solution and for stable double-hexamer formation.
  • CDK phosphorylation of ORC disrupts the Orc6 tethering interaction, inhibiting Mcm2-7 double-hexamer formation.
  • Mutations in the Orc6 linker region can support MO complex formation but impair subsequent Mcm2-7 recruitment.

Conclusions:

  • Orc6 plays a critical role in multiple stages of origin licensing, acting as a molecular tether during the one-ORC helicase loading mechanism.
  • The Orc6-Mcm2 interaction provides a molecular explanation for efficient and sequential loading of Mcm2-7 double hexamers.
  • Regulation of the Orc6 tethering interaction by CDK phosphorylation offers insight into cell cycle control of DNA replication initiation.