Plaza-G A Ismael, Ortiz-Rodríguez María, Buchanan Seth P, Miguez-Amil Samuel, Lemishko Kateryna M, Moreno-Herrero Fernando, Fernandez-Leiro Rafael, Ciesielski Grzegorz L, Ibarra Borja
Instituto Madrileño de Estudios Avanzados en Nanociencia, IMDEA Nanociencia, Madrid, Spain.
Department of Biology, University of North Florida, Jacksonville, FL, USA.
Nat Commun. 2025 Jul 1;16(1):5460. doi: 10.1038/s41467-025-60289-0.
The human mitochondrial helicase Twinkle is essential for mitochondrial DNA (mtDNA) replication and integrity. Using biochemical and single-molecule techniques, we investigated Twinkle's real-time kinetics, including DNA loading, unwinding, and rewinding, and their regulation by its N-terminal Zinc-binding domain (ZBD), C-terminal tail, and mitochondrial SSB protein (mtSSB). Our results indicate that Twinkle rapidly scans dsDNA to locate the fork, where specific interactions halt diffusion. During unwinding, ZBD-DNA interactions and C-terminal tail control of ATPase activity downregulate kinetics, slowing down the helicase. Binding of mtSSB to DNA likely outcompetes ZBD-DNA interactions, alleviating the downregulatory effects of this domain. Furthermore, we show that ZBD-DNA interactions and ATP binding also regulate rewinding kinetics following helicase stalling. Our findings reveal that ZBD and C-terminal tail play a major role in regulation of Twinkle´s real-time kinetics. Their interplay constitutes an auto-regulatory mechanism that may be relevant for coordinating the mtDNA maintenance activities of the helicase.
人类线粒体解旋酶Twinkle对于线粒体DNA（mtDNA）的复制和完整性至关重要。我们运用生化和单分子技术，研究了Twinkle的实时动力学，包括DNA加载、解旋和重新缠绕，以及其N端锌结合结构域（ZBD）、C端尾巴和线粒体单链结合蛋白（mtSSB）对这些过程的调控。我们的结果表明，Twinkle会快速扫描双链DNA以定位叉状结构，在那里特定的相互作用会阻止扩散。在解旋过程中，ZBD与DNA的相互作用以及C端尾巴对ATP酶活性的控制会下调动力学，使解旋酶的速度减慢。mtSSB与DNA的结合可能会胜过ZBD与DNA的相互作用，从而减轻该结构域的下调作用。此外，我们还表明，ZBD与DNA的相互作用以及ATP结合在解旋酶停滞后的重新缠绕动力学中也起到调控作用。我们的研究结果揭示，ZBD和C端尾巴在调控Twinkle的实时动力学中发挥着主要作用。它们之间的相互作用构成了一种自动调节机制，这可能与协调解旋酶的mtDNA维持活动相关。
