Autoregulation of the real-time kinetics of the human mitochondrial replicative helicase

Ismael Plaza-G A, María Ortiz-Rodríguez, Seth P. Buchanan, Samuel Miguez-Amil, Kateryna M. Lemishko, Fernando Moreno-Herrero, Rafael Fernandez-Leiro, Grzegorz L. Ciesielski () and Borja Ibarra ()
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Ismael Plaza-G A: IMDEA Nanociencia
María Ortiz-Rodríguez: IMDEA Nanociencia
Seth P. Buchanan: University of North Florida
Samuel Miguez-Amil: Spanish National Cancer Research Centre (CNIO)
Kateryna M. Lemishko: IMDEA Nanociencia
Fernando Moreno-Herrero: Centro Nacional de Biotecnología (CSIC)
Rafael Fernandez-Leiro: Spanish National Cancer Research Centre (CNIO)
Grzegorz L. Ciesielski: University of North Florida
Borja Ibarra: IMDEA Nanociencia

Nature Communications, 2025, vol. 16, issue 1, 1-13

Abstract: Abstract 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.

Date: 2025
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DOI: 10.1038/s41467-025-60289-0

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