Speaker
Description
Mitochondrial DNA (mtDNA) integrity is essential for proper cellular function and relies on the coordinated activities of several mitochondrial proteins. In this study, we employed two single-molecule fluorescence-based approaches—single-molecule Förster resonance energy transfer (smFRET) and nuclease-induced stepwise photodropping (NISP)—to investigate the mechanistic roles of the mitochondrial helicase TWINKLE, the mitochondrial single-stranded DNA-binding protein (mtSSB), and the single-stranded DNA-specific exonuclease MGME1 in real time.
Using smFRET, we observed that TWINKLE exhibits occasional pausing behavior, likely arising from its stochastic ATP hydrolysis dynamics. We further found that mtSSB stabilizes the TWINKLE–DNA interaction and reduces fraying or futile reannealing events, thereby enhancing unwinding efficiency. In addition, the NISP assay enabled us to quantify the directional exonuclease activity of MGME1, revealing degradation rates of 6.3 ± 0.4 nt s⁻¹ in the 5′→3′ direction and 2.0 ± 0.1 nt s⁻¹ in the 3′→5′ direction. These results provide direct evidence for MGME1’s preferential 5′ directionality, consistent with its established function in mtDNA maintenance.
Together, these findings demonstrate the utility of integrated single-molecule assays for dissecting the molecular mechanisms underlying mitochondrial genome integrity. Future work combining smFRET and NISP will allow detailed investigation of the coordinated actions and interplay among mitochondrial replication and repair proteins at the single-molecule level.
