Autoregulation of the real-time kinetics of the human mitochondrial replicative helicase
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-termi...
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Nature Portfolio
2025-07-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-60289-0 |
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| author | 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 Borja Ibarra |
| author_facet | 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 Borja Ibarra |
| author_sort | Ismael Plaza-G A |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-75491ed2c0744f65ba194df2b4efbe8d |
| institution | Kabale University |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Nature Portfolio |
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| series | Nature Communications |
| spelling | doaj-art-75491ed2c0744f65ba194df2b4efbe8d2025-08-20T04:01:35ZengNature PortfolioNature Communications2041-17232025-07-0116111310.1038/s41467-025-60289-0Autoregulation of the real-time kinetics of the human mitochondrial replicative helicaseIsmael Plaza-G A0María Ortiz-Rodríguez1Seth P. Buchanan2Samuel Miguez-Amil3Kateryna M. Lemishko4Fernando Moreno-Herrero5Rafael Fernandez-Leiro6Grzegorz L. Ciesielski7Borja Ibarra8Instituto Madrileño de Estudios Avanzados en Nanociencia, IMDEA NanocienciaInstituto Madrileño de Estudios Avanzados en Nanociencia, IMDEA NanocienciaDepartment of Biology, University of North FloridaStructural Biology Programme, Spanish National Cancer Research Centre (CNIO)Instituto Madrileño de Estudios Avanzados en Nanociencia, IMDEA NanocienciaCentro Nacional de Biotecnología (CSIC)Structural Biology Programme, Spanish National Cancer Research Centre (CNIO)Department of Biology, University of North FloridaInstituto Madrileño de Estudios Avanzados en Nanociencia, IMDEA NanocienciaAbstract 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.https://doi.org/10.1038/s41467-025-60289-0 |
| spellingShingle | 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 Borja Ibarra Autoregulation of the real-time kinetics of the human mitochondrial replicative helicase Nature Communications |
| title | Autoregulation of the real-time kinetics of the human mitochondrial replicative helicase |
| title_full | Autoregulation of the real-time kinetics of the human mitochondrial replicative helicase |
| title_fullStr | Autoregulation of the real-time kinetics of the human mitochondrial replicative helicase |
| title_full_unstemmed | Autoregulation of the real-time kinetics of the human mitochondrial replicative helicase |
| title_short | Autoregulation of the real-time kinetics of the human mitochondrial replicative helicase |
| title_sort | autoregulation of the real time kinetics of the human mitochondrial replicative helicase |
| url | https://doi.org/10.1038/s41467-025-60289-0 |
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