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

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.