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

Area of Science:

• Molecular Biology
• DNA Replication
• Chromatin Biology

Background:

• Bidirectional DNA replication requires the head-to-head loading of two Mcm2-7 helicase complexes at replication origins.
• The helicase loader ORC (Origin Recognition Complex) is responsible for sequentially loading these Mcm2-7 hexamers.
• ORC must switch from a high-affinity DNA binding site to a weaker, inverted site for proper helicase orientation, but the mechanism is unclear.

Approach:

• Utilized single-molecule Förster resonance energy transfer (sm-FRET) to monitor dynamic interactions between DNA, ORC, and Mcm2-7.
• Investigated the role of DNA bending and protein dissociation in facilitating ORC site switching.
• Analyzed the composition and dynamics of sliding helicase-loading intermediates, including ORC, Mcm2-7, and Cdt1.

Key Points:

• Loss of DNA bending during Mcm2-7 deposition accelerates ORC dissociation from DNA.
• Temporally-controlled DNA sliding of ORC-Mcm2-7-Cdt1 complexes occurs during helicase loading.
• Sequential DNA unbending, Cdc6 release, and sliding progressively destabilize ORC binding, enabling site switching.

Conclusions:

• A stepwise decrease in ORC DNA-binding stability facilitates its dissociation and reorientation for sequential Mcm2-7 loading.
• Dynamic protein-DNA interactions, including DNA unbending and controlled sliding, are crucial for ORC site switching.
• This mechanism ensures the correct loading of oppositely-oriented Mcm2-7 helicases, essential for bidirectional DNA replication initiation.