Wang Hsin-Yi, Lee Ying-Ying, Chien Po-Jung, Tsai Wen-An, Sun Pei-Jia, Wang Li-Ting, Wu Chyuan-Chuan, Fan Hsiu-Fang
Institute of Medical Science and Technology, National Sun Yat-sen University, Kaohsiung, 804, Taiwan.
Department of Chemistry, National Sun Yat-sen University, Kaohsiung, 804, Taiwan.
Nucleic Acids Res. 2025 Aug 27;53(16). doi: 10.1093/nar/gkaf803.
The mitochondrial DNA helicase TWINKLE, a hexameric ring-shaped helicase, plays a crucial role in maintaining mitochondrial DNA integrity. TWINKLE translocates along one DNA strand, unwinding the duplex by excluding the complementary strand through coordinated ATP hydrolysis. However, the precise mechanisms underlying this process remain incompletely understood. In this study, we utilized single-molecule Förster Resonance Energy Transfer (smFRET) to investigate the mechanisms of TWINKLE-mediated DNA unwinding. Our results reveal that TWINKLE occasionally pauses during unwinding, with the rate of unwinding and the duration of pausing strongly influenced by ATP concentration, but not by the presence of DNA mismatches or mitochondrial single-stranded DNA-binding protein (mtSSB). These findings suggest that the pausing events primarily arise from stochastic ATP hydrolysis within the helicase subunits. DNA mismatches exacerbate TWINKLE's pausing and dissociation from DNA, thereby impairing DNA unwinding. In contrast, mtSSB significantly mitigates helicase dissociation by stabilizing TWINKLE-DNA interactions. This study provides novel insights into the functional dynamics of TWINKLE, highlighting the role of ATP hydrolysis in orchestrating single-stranded DNA translocation, the detrimental effects of DNA mismatches on DNA unwinding, and the critical role of mtSSB in supporting helicase function.
线粒体DNA解旋酶TWINKLE是一种六聚体环状解旋酶，在维持线粒体DNA完整性方面发挥着关键作用。TWINKLE沿着一条DNA链移位，通过协调ATP水解排除互补链来解开双链。然而，这一过程背后的确切机制仍未完全了解。在本研究中，我们利用单分子荧光共振能量转移（smFRET）来研究TWINKLE介导的DNA解旋机制。我们的结果表明，TWINKLE在解旋过程中偶尔会暂停，解旋速率和暂停持续时间受ATP浓度的强烈影响，但不受DNA错配或线粒体单链DNA结合蛋白（mtSSB）的存在影响。这些发现表明，暂停事件主要源于解旋酶亚基内的随机ATP水解。DNA错配会加剧TWINKLE的暂停和与DNA的解离，从而损害DNA解旋。相比之下，mtSSB通过稳定TWINKLE-DNA相互作用显著减轻解旋酶的解离。本研究为TWINKLE的功能动力学提供了新的见解，突出了ATP水解在协调单链DNA移位中的作用、DNA错配对DNA解旋的有害影响以及mtSSB在支持解旋酶功能中的关键作用。
