Cryo-EM structure of a licensed DNA replication origin

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Eukaryotic DNA replication begins at origins, requiring MCM helicase loading.
• The MCM helicase encircles duplex DNA post-ATP hydrolysis, forming an inactive double hexamer.
• Origin firing involves MCM engagement with Cdc45 and GINS to form the CMG holo-helicase, a process requiring MCM phosphorylation by DDK.

Purpose of the Study:

• To determine the cryo-EM structures of DNA-bound MCM, both unmodified and phosphorylated.
• To visualize phospho-dependent MCM elements crucial for Cdc45 recruitment.
• To understand the conformational transition from MCM to CMG and its role in DNA unwinding.

Main Methods:

• Cryo-electron microscopy (cryo-EM) to resolve structures of DNA-bound MCM.
• Structural comparison between MCM-DNA and CMG-DNA complexes.

Main Results:

• Determined cryo-EM structures of DNA-bound MCM (unmodified and phosphorylated).
• Identified a phospho-dependent MCM element potentially involved in Cdc45 recruitment.
• Observed MCM pore loops constraining duplex DNA in a bent conformation.

Conclusions:

• The MCM-DNA structure provides insights into the loaded, post-catalytic state of the helicase.
• The transition to CMG likely involves conformational changes promoting DNA untwisting and melting.
• These findings advance our understanding of eukaryotic origin activation and replication initiation.