BioSciences Department, Rice University, Houston, TX 77005, USA.
Physics Department, North Carolina State University, Raleigh, NC 27695, USA.
Nucleic Acids Res. 2022 Nov 11;50(20):11965-11978. doi: 10.1093/nar/gkac1089.
Twinkle is a mitochondrial replicative helicase which can self-load 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 microscopy (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 important role of the non-catalytic N-terminal domain of Twinkle. The N-terminal domain directly contacts the C-terminal helicase domain, and the contact interface is a hotspot for disease-related mutations. Mutations at the interface destabilize Twinkle hexamer and reduce helicase activity. With HS-AFM, we observed that a highly dynamic Twinkle domain, which is likely to be the N-terminal domain, can protrude ∼5 nm to transiently capture nearby DNA and initialize Twinkle loading onto DNA. Moreover, structural analysis and subunit doping experiments suggest that Twinkle hydrolyzes ATP stochastically, which is distinct from related helicases from bacteriophages.
Twinkle 是一种线粒体复制解旋酶，能够自我加载并解开线粒体 DNA。近 60 种 Twinkle 突变与人类线粒体疾病有关。我们使用冷冻电子显微镜（cryo-EM）和高速原子力显微镜（HS-AFM）获得了具有 DNA 的脊椎动物 Twinkle 同源物的原子分辨率结构，并实时捕获了 Twinkle 如何自我加载到 DNA 上。我们的数据突出了 Twinkle 非催化 N 端结构域的重要作用。N 端结构域直接与 C 端解旋酶结构域接触，接触界面是与疾病相关突变的热点。界面处的突变会使 Twinkle 六聚体失稳并降低解旋酶活性。通过 HS-AFM，我们观察到一个高度动态的 Twinkle 结构域（可能是 N 端结构域）可以向外突出约 5nm，以瞬时捕获附近的 DNA 并启动 Twinkle 加载到 DNA 上。此外，结构分析和亚基掺杂实验表明，Twinkle 随机水解 ATP，这与噬菌体的相关解旋酶不同。
