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Changing protein-DNA interactions promote ORC binding-site exchange during replication origin licensing.

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|July 18, 2023
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Summary

The origin recognition complex (ORC) loads two Mcm2-7 helicases for DNA replication by switching binding sites. DNA unbending and sliding facilitate ORC release, enabling sequential helicase loading and bidirectional replication.

Keywords:
DNA replication initiationMcm2-7 helicaseorigin licensingorigin recognition complex (ORC)single-molecule FRET

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Eukaryotic DNA replication initiation requires the Mcm2-7 helicase, loaded by the origin recognition complex (ORC).
  • ORC sequentially loads two Mcm2-7 hexamers in a head-to-head orientation to prime origins for bidirectional replication.
  • ORC must transition from a high-affinity to a lower-affinity DNA binding site to achieve this sequential loading, but the mechanism is unclear.

Purpose of the Study:

  • To elucidate the mechanism by which ORC switches DNA binding sites during the sequential loading of Mcm2-7 helicases.
  • To investigate the dynamic interactions between DNA, ORC, and Mcm2-7 during origin licensing.

Main Methods:

  • Single-molecule Förster resonance energy transfer (smFRET) was employed to monitor DNA-protein interactions.
  • Studies focused on the changing interactions between DNA and ORC or Mcm2-7 during the loading process.

Main Results:

  • Loss of DNA bending during Mcm2-7 deposition accelerates ORC dissociation from DNA.
  • Temporally controlled DNA sliding of intermediate complexes, including ORC, Mcm2-7, and Cdt1, was observed.
  • Sequential DNA unbending, Cdc6 release, and DNA sliding progressively decrease ORC's DNA binding stability, facilitating site switching.

Conclusions:

  • Dynamic protein-DNA interactions, including DNA unbending and sliding, are crucial for ORC-mediated sequential loading of Mcm2-7 helicases.
  • This mechanism ensures the correct head-to-head alignment of helicases for efficient bidirectional DNA replication.
  • The observed controlled sliding provides insights into how ORC accesses secondary DNA binding sites.