Structural and dynamic basis of DNA capture and translocation by mitochondrial Twinkle helicase

Area of Science:

• Molecular Biology
• Structural Biology
• Genetics

Background:

• Twinkle is a mitochondrial replicative helicase essential for mitochondrial DNA maintenance.
• Mutations in Twinkle are associated with various human mitochondrial diseases.
• Understanding Twinkle's structure and function is critical for elucidating disease mechanisms.

Purpose of the Study:

• To determine the atomic-resolution structure of a vertebrate Twinkle homolog bound to DNA.
• To visualize the real-time process of Twinkle self-loading onto DNA.
• To investigate the role of the N-terminal domain in Twinkle's function and disease association.

Main Methods:

• Cryo-electron microscopy (cryo-EM) for high-resolution structural determination.
• High-speed atomic force microscopy (HS-AFM) for real-time dynamic observations.
• Structural analysis and subunit doping experiments for functional insights.

Main Results:

• Obtained the atomic-resolution structure of Twinkle with DNA, revealing the N-terminal domain's interaction with the C-terminal helicase domain.
• Identified the N-terminal/C-terminal interface as a hotspot for disease-related mutations that destabilize the hexamer and reduce helicase activity.
• Observed dynamic N-terminal domain protrusion facilitating DNA capture and Twinkle loading via HS-AFM.
• Demonstrated stochastic ATP hydrolysis by Twinkle, differentiating it from related helicases.

Conclusions:

• The N-terminal domain plays a critical role in Twinkle's self-loading mechanism and overall stability.
• Disease-associated mutations cluster at the interface, disrupting hexamer stability and helicase function.
• Twinkle exhibits unique stochastic ATP hydrolysis, suggesting distinct functional regulation compared to other helicases.