Cryo-EM structures of the eukaryotic replicative helicase bound to a translocation substrate

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• The Cdc45-MCM-GINS (CMG) helicase is essential for eukaryotic DNA replication.
• Its function relies on the MCM ATPase activity for DNA unwinding.
• Mechanisms of CMG translocation and ATP-driven unwinding are not fully understood.

Purpose of the Study:

• To elucidate the structural basis of CMG helicase translocation on DNA forks.
• To investigate how ATP hydrolysis by MCM drives DNA unwinding at the replication fork.

Main Methods:

• Cryo-electron microscopy (cryo-EM) to determine high-resolution structures of CMG on DNA forks.
• Single-molecule Förster Resonance Energy Transfer (smFRET) measurements to support structural findings.

Main Results:

• Two distinct CMG structures on DNA forks were resolved at subnanometre resolution.
• In one state, the MCM ATPase module contacts single-stranded DNA via specific hairpins.
• In another state, the MCM motor appears relaxed, with DNA interacting with the N-terminal tier.

Conclusions:

• A model for replication fork unwinding is proposed, involving concerted action of MCM's N-terminal and AAA+ tiers.
• CMG translocation likely occurs on single-stranded DNA.
• These findings provide mechanistic insights into eukaryotic genome duplication.