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Review
. 2019 Nov 26;58(47):4667-4676.
doi: 10.1021/acs.biochem.9b00346. Epub 2019 Jun 28.

A Tour de Force on the Double Helix: Exploiting DNA Mechanics To Study DNA-Based Molecular Machines

Michael R Wasserman  1 , Shixin Liu  1
Affiliations
Review

A Tour de Force on the Double Helix: Exploiting DNA Mechanics To Study DNA-Based Molecular Machines

Michael R Wasserman et al. Biochemistry. .

Abstract

DNA is both a fundamental building block of life and a fascinating natural polymer. The advent of single-molecule manipulation tools made it possible to exert controlled force on individual DNA molecules and measure their mechanical response. Such investigations elucidated the elastic properties of DNA and revealed its distinctive structural configurations across force regimes. In the meantime, a detailed understanding of DNA mechanics laid the groundwork for single-molecule studies of DNA-binding proteins and DNA-processing enzymes that bend, stretch, and twist DNA. These studies shed new light on the metabolism and transactions of nucleic acids, which constitute a major part of the cell's operating system. Furthermore, the marriage of single-molecule fluorescence visualization and force manipulation has enabled researchers to directly correlate the applied tension to changes in the DNA structure and the behavior of DNA-templated complexes. Overall, experimental exploitation of DNA mechanics has been and will continue to be a unique and powerful strategy for understanding how molecular machineries recognize and modify the physical state of DNA to accomplish their biological functions.

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Figures

Figure 1.
Figure 1.
Single-molecule manipulation to elucidate DNA mechanics (A) Force measurements of single DNA molecules are enabled via multiple optical-trapping geometries, in which one end of the DNA is tethered to an optically trapped micron-sized bead, and the other end is tethered to a microscope slide surface (Top), a micropipette-suctioned bead (Middle), or another optically trapped bead (Bottom). Figure reprinted with permission from. Copyright (2014) American Chemical Society. (B) Force-extension behavior of ssDNA and dsDNA. The red lines indicate the WLC model prediction. At forces above the crossover point (~6 pN), ssDNA is longer than dsDNA. Figure reproduced with permission from. (C) Force-torque phase diagram of dsDNA. Note that along the borders between regions, adjacent phases coexist in equilibrium. Z: Z-DNA; S: S-DNA; B: B-DNA; P: P-DNA (extended and overtwisted); Sc-P: plectonemically supercoiled DNA. Figure adapted with permission from. Copyright (2001) American Physical Society.
Figure 2.
Figure 2.
Optical-trapping assay to study viral DNA packaging (A) Schematic of a dual-trap optical tweezers assay to study DNA translocation by the bacteriophage φ29 packaging motor. Figure reproduced with permission from. (B) (Top) Representative packaging traces collected at low (Left) and high force (Right). At high force, the 10-bp bursts seen at low force are decelerated enough to reveal 2.5-bp steps. (Bottom) Pairwise distance analysis for the corresponding traces. Figure reproduced with permission from. (C) Mechanochemical model for the φ29 packaging motor showing DNA translocation cycles with a dwell-burst structure. Figure adapted with permission from.
Figure 3.
Figure 3.
Flow-stretching assay to study DNA replication (A) Schematic of a DNA flow-stretching assay. Individual DNA molecules are tethered to the surface of a flow cell on one end and conjugated to a bead on the other end. Figure reproduced with permission from. (B) (Top) Representative single-molecule trajectories with and without ribonucleotides required for lagging-strand priming. Arrows indicate pausing events. (Bottom) Schematic depicting that leading-strand synthesis causes conversion of the 5’ tail of the tethered strand from dsDNA to ssDNA, resulting in a decrease in the tether length. Figure reproduced with permission from, originally adapted with permission from.
Figure 4.
Figure 4.
DNA structural transitions revealed by combined fluorescence-force microscopy (A) Representative force-extension curve for dsDNA tethered to optically trapped beads via the 3’ end of each strand. (B) Fluorescence images of a dsDNA molecule tethered by the geometry depicted in (A) and stained with the intercalating dye YOYO. At tensions resulting in tether lengths greater than the contour length (L0), unstained regions emerged, signifying the overstretching transition. (C) Representative force-extension curve for dsDNA tethered to beads via both ends of each strand. Here the overstretching transition occurs at ~110 pN. (D) Cartoon representations of various structural forms when dsDNA is overstretched. (E) Fluorescence images of an overstretched DNA that is topologically closed but torsionally relaxed. At low ionic strength, melting bubbles are visualized with fluorescent RPA. dsDNA regions are indicated by the intercalating dye Sytox. (F) Fluorescence images of an overstretched and nicked DNA. Here strand unpeeling is favored, as indicated by the orange arrows. Panels A-D are reproduced with permission from. Panels E and F are reproduced with permission from.
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