Structural and Dynamic Basis of DNA Capture and Translocation by Mitochondrial Twinkle Helicase

ABSTRACT Twinkle is the sole helicase responsible for mitochondrial DNA replication. Twinkle can selfload onto and unwind mitochondrial DNA. Nearly 60 mutations on Twinkle have been linked to human mitochondrial diseases. Using cryo-electron microscopy (cryo-EM) and High-speed atomic force microscope (HS-AFM), we obtained the atomic-resolution structure of a vertebrate Twinkle homolog with DNA and captured in real-time how Twinkle is self-loaded onto DNA. Our data highlight the essential role of the non-catalytic N-terminal domain of Twinkle. The N-terminal domain interacts with the helicase domain to stabilize Twinkle hexamers during translocation and the new domain-domain interface is a hotspot for disease-related mutations. The N-terminal domains can protrude approximately 5 nm to capture nearby DNA and initialize Twinkle loading onto DNA. Moreover, structural analysis and subunit doping experiments suggest that Twinkle hydrolyzes ATP stochastically, which explains the low efficiency of Twinkle DNA unwinding and implicates additional regulations of Twinkle during mitochondrial DNA replication.