Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 295, Issue 17, Pages 5564-5576Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA120.012795
Keywords
atomic force microscopy (AFM); DNA binding protein; DNA helicase; DNA replication; mitochondrial disease; mitochondrial DNA (mtDNA); single-molecule biophysics; ssDNA-binding protein (SSB); structural dynamics; Twinkle helicase
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Funding
- Extramural Research Program of the National Institutes of Health [R01GM107559, R21ES027641, R01GM123246]
- North Carolina State University Center for Human Health and the Environment Pilot Project [P30ES025128]
- Intramural Research Program of the NIEHS, National Institutes of Health [ES065078]
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Knowledge of the molecular events in mitochondrial DNA (mtDNA) replication is crucial to understanding the origins of human disorders arising from mitochondrial dysfunction. Twinkle helicase is an essential component of mtDNA replication. Here, we employed atomic force microscopy imaging in air and liquids to visualize ring assembly, DNA binding, and unwinding activity of individual Twinkle hexamers at the single-molecule level. We observed that the Twinkle subunits self-assemble into hexamers and higher-order complexes that can switch between open and closed-ring configurations in the absence of DNA. Our analyses helped visualize Twinkle loading onto and unloading from DNA in an open-ringed configuration. They also revealed that closed-ring conformers bind and unwind several hundred base pairs of duplex DNA at an average rate of ?240 bp/min. We found that the addition of mitochondrial single-stranded (ss) DNA?binding protein both influences the ways Twinkle loads onto defined DNA substrates and stabilizes the unwound ssDNA product, resulting in a ?5-fold stimulation of the apparent DNA-unwinding rate. Mitochondrial ssDNA-binding protein also increased the estimated translocation processivity from 1750 to >9000 bp before helicase disassociation, suggesting that more than half of the mitochondrial genome could be unwound by Twinkle during a single DNA-binding event. The strategies used in this work provide a new platform to examine Twinkle disease variants and the core mtDNA replication machinery. They also offer an enhanced framework to investigate molecular mechanisms underlying deletion and depletion of the mitochondrial genome as observed in mitochondrial diseases.
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