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Review
. 2010 Jun 18;285(25):18967-71.
doi: 10.1074/jbc.R109.092437. Epub 2010 Apr 9.

Single-molecule measurements of DNA topology and topoisomerases

Keir C Neuman  1
Affiliations
Review

Single-molecule measurements of DNA topology and topoisomerases

Keir C Neuman. J Biol Chem. .

Abstract

Topological properties of DNA influence its mechanical and biochemical interactions. Genomic DNA is maintained in a state of topological homeostasis by topoisomerases and is subjected to mechanical stress arising from replication and segregation. Despite their fundamental roles, the effects of topology and force have been difficult to ascertain. Developments in single-molecule manipulation techniques have enabled precise control and measurement of the topology of individual DNA molecules under tension. This minireview provides an overview of these single-molecule techniques and illustrates their unique capabilities through a number of specific examples of single-molecule measurements of DNA topology and topoisomerase activity.

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Figures

FIGURE 1.
FIGURE 1.
Single-molecule techniques to control DNA topology (not to scale). a, magnetic tweezers consist of two magnets (red and blue) held above a microscope flow cell in which a magnetic bead (blue) is tethered to the surface (light blue) by a single DNA molecule (red and blue). The magnets impose an upward force on the beads, which depends on the vertical position of the magnets (black arrow). Rotating the magnets (black curved arrow) rotates the bead (red curved arrow), thereby changing the linking number of the DNA. b, optical rotation uses a focused laser (pink) to apply force on a quartz cylinder (blue) that is tethered to the surface of the flow cell by a molecule of DNA. The optical axis of the cylinder (blue arrow) experiences a torque that tends to align it with the polarization direction of the laser (pink arrow), which is focused by a microscope objective below the flow cell. c, rotor bead tracking uses a reporter bead (green) attached near a specific nick in the DNA molecule (red star). A magnetic bead (blue) at the distal end of the DNA allows torque and tension to be applied.
FIGURE 2.
FIGURE 2.
Single-molecule measurements of DNA topology and topoisomerase activity. a, extension-rotation (ΔLk) relationship for a 3-kb DNA molecule (Y. Seol and K. C. Neuman, unpublished data). At low force (0.4 pN; red circles), the extension decreases symmetrically as the linking number is changed from Lk0. At ∼5 turns, the DNA buckles, and subsequent turns increase the number of plectoneme crossings (inset schematics) and decrease the extension. At higher force (1.6 pN; green circles), the extension for positive supercoiling is similar, although the buckling transition occurs at ∼11 turns. However, for negative supercoiling, the DNA melts to form locally unwound regions (schematic to the left). b, single-molecule measurement of supercoil relaxation by topoisomerase IV (adapted from Ref. 34). At time 0, the DNA molecule is negatively supercoiled, decreasing Wr by 8 though the introduction of eight right-handed crossings, which decreases the extension from the relaxed length of ∼0.7 μm. Individual relaxation events are observed as abrupt increases in extension (arrows).
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