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. 2021 Sep 5;11(17):e4139.
doi: 10.21769/BioProtoc.4139.

Using Atomic Force Microscopy to Study the Real Time Dynamics of DNA Unwinding by Mitochondrial Twinkle Helicase

Parminder Kaur  1   2 , Hai Pan  1 , Matthew J Longley  3 , William C Copeland  3 , Hong Wang  1   2   4
Affiliations

Using Atomic Force Microscopy to Study the Real Time Dynamics of DNA Unwinding by Mitochondrial Twinkle Helicase

Parminder Kaur et al. Bio Protoc. .

Abstract

Understanding the structure and dynamics of DNA-protein interactions during DNA replication is crucial for elucidating the origins of disorders arising from its dysfunction. In this study, we employed Atomic Force Microscopy as a single-molecule imaging tool to examine the mitochondrial DNA helicase Twinkle and its interactions with DNA. We used imaging in air and time-lapse imaging in liquids to observe the DNA binding and unwinding activities of Twinkle hexamers at the single-molecule level. These procedures helped us visualize Twinkle loading onto and unloading from the DNA in the open-ring conformation. Using traditional methods, it has been shown that Twinkle is capable of unwinding dsDNA up to 20-55 bps. We found that the addition of mitochondrial single-stranded DNA binding protein (mtSSB) facilitates a 5-fold increase in the DNA unwinding rate for the Twinkle helicase. The protocols developed in this study provide new platforms to examine DNA replication and to explore the mechanism driving DNA deletion and human diseases. Graphic abstract: Mitochondrial Twinkle Helicase Dynamics.

Keywords: Atomic Force Microscope; Liquid AFM imaging; Mitochondria; Mitochondrial replication; Single molecule imaging; Twinkle helicase.

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Conflict of interest statement

Competing interestsThe authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.. SPI Chem Mica used for Atomic Force Microscopy (AFM) experiments
Figure 2.
Figure 2.. AFM instrument details showing basic AFM components inside the AFM noise canceling chamber with the AC2 controller
Figure 3.
Figure 3.. AFM head with labeled controls
Figure 4.
Figure 4.. Mode Master tab with AC air topography mode selected (red square)
Figure 5.
Figure 5.. Labeled cantilever holder parts with mounted AFM tip
Figure 6.
Figure 6.. Cantilever tune and Tune Panel tab of the Master Panel showing target amplitude of 1 V selected (red square)
Figure 7.
Figure 7.. Main tab and Advanced Scan Parms tab of the Master Panel showing Feedback filter at 1.5 kHz (red square) in AAC air imaging mode
Figure 8.
Figure 8.. Mode Master tab with AC water Topography mode selected (red square)
Figure 9.
Figure 9.. Main tab and Advanced Scan Parms tab of the Master Panel showing Thermal tab and Feedback filter at 0.5 kHz (red square) in AAC water imaging mode
Figure 10.
Figure 10.. Height measurement steps of a (A) DNA AFM image (B) with its cross-sectional analysis showing a height measured in the (C) analyze panel (dZ) (red square)
Figure 11.
Figure 11.. Contour length measurement steps of a (A) traced DNA AFM image (B) with its cross-sectional analysis showing a height measured in the (C) analyze panel (dXY) (red square)
See this image and copyright information in PMC

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