Molecular Basis for ATP-Hydrolysis-Driven DNA Translocation by the CMG Helicase of the Eukaryotic Replisome

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• The eukaryotic replisome utilizes the CMG (Cdc45-MCM-GINS) helicase for DNA unwinding.
• The hexameric MCM (minichromosome maintenance) ATPase is central to CMG function, but its precise unwinding mechanism remains unclear.
• The asymmetric nature of ATPase site requirements within the MCM ring suggests a complex, non-uniform mechanism.

Purpose of the Study:

• To elucidate the ATP-hydrolysis-driven translocation mechanism of the CMG helicase.
• To investigate the role of the MCM ring's ATPase sites in DNA unwinding.
• To examine the interaction between the CMG helicase and fork-stabilizing complexes within a higher-order replisome assembly.

Main Methods:

• Cryo-electron microscopy (cryo-EM) was employed to image the CMG helicase during ATP-hydrolysis-driven translocation.
• High-resolution structural analysis of different helicase states was performed.
• Imaging of a higher-order replisome assembly including the Mrc1-Csm3-Tof1 complex was conducted.

Main Results:

• The CMG helicase engages DNA using four neighboring MCM protomers at a time, with ATP binding facilitating DNA interaction.
• A non-symmetric hand-over-hand rotary mechanism is proposed, explaining the asymmetric ATPase requirements within the MCM ring.
• The Mrc1-Csm3-Tof1 complex enhances the interaction between parental DNA and the CMG at the replication fork.

Conclusions:

• The study reveals a novel hand-over-hand rotary mechanism for CMG helicase-mediated DNA unwinding.
• The findings explain the functional asymmetry of the MCM ATPase sites.
• The Mrc1-Csm3-Tof1 complex likely couples DNA translocation with fork unwinding, ensuring replication fidelity.