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The impact of DNA intercalators on DNA and DNA-processing enzymes elucidated through force-dependent binding kinetics.

Biebricher AS, Heller I, Roijmans RF, Hoekstra TP, Peterman EJ, Wuite GJ - Nat Commun (2015)

Bottom Line: Here we elucidate this perturbation by combining single-dye fluorescence microscopy with force spectroscopy and measuring the kinetics of DNA intercalation by the mono- and bis-intercalating cyanine dyes SYTOX Orange, SYTOX Green, SYBR Gold, YO-PRO-1, YOYO-1 and POPO-3.These rates can be tuned over a range of seven orders of magnitude by changing DNA tension, intercalating species and ionic strength.We show that optimizing these rates minimizes the impact of intercalators on strand separation and enzymatic activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics and Astronomy, LaserLaB Amsterdam, VU University Amsterdam, De Boelelaan 1081, Amsterdam 1081HV, The Netherlands.

ABSTRACT
DNA intercalators are widely used as fluorescent probes to visualize DNA and DNA transactions in vivo and in vitro. It is well known that they perturb DNA structure and stability, which can in turn influence DNA-processing by proteins. Here we elucidate this perturbation by combining single-dye fluorescence microscopy with force spectroscopy and measuring the kinetics of DNA intercalation by the mono- and bis-intercalating cyanine dyes SYTOX Orange, SYTOX Green, SYBR Gold, YO-PRO-1, YOYO-1 and POPO-3. We show that their DNA-binding affinity is mainly governed by a strongly tension-dependent dissociation rate. These rates can be tuned over a range of seven orders of magnitude by changing DNA tension, intercalating species and ionic strength. We show that optimizing these rates minimizes the impact of intercalators on strand separation and enzymatic activity. These new insights provide handles for the improved use of intercalators as DNA probes with minimal perturbation and maximal efficacy.

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Impact of intercalators on DNA overstretching and DNA-enzyme activity.(a–c) Kymographs and corresponding force-extension curves (red) in the presence of YOYO (a), SbG (b) and YO (c) at 100 mM NaCl. The kymograph image and corresponding force-extension curves are co-aligned along the horizontal axis, which is possible because of the constant stretching speed of the moving bead (visible as the bright, upward tilted bar in the image). Thus, the fluorescence pattern in the vertical direction in the kymograph is a DNA-staining pattern at the DNA extension indicated on the horizontal axis. Cyan curves, no intercalator present. (d) Time traces recorded at 40 pN showing dsDNA digestion by the exonuclease activity of T7 DNA polymerase. Black, no intercalator; red, YO; blue, SxO; green, SbG. Under these experimental conditions, YOYO abolished all exonuclease activities. (e) Histograms of the resulting average digestion rates obtained at the four different conditions (Methods).
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f7: Impact of intercalators on DNA overstretching and DNA-enzyme activity.(a–c) Kymographs and corresponding force-extension curves (red) in the presence of YOYO (a), SbG (b) and YO (c) at 100 mM NaCl. The kymograph image and corresponding force-extension curves are co-aligned along the horizontal axis, which is possible because of the constant stretching speed of the moving bead (visible as the bright, upward tilted bar in the image). Thus, the fluorescence pattern in the vertical direction in the kymograph is a DNA-staining pattern at the DNA extension indicated on the horizontal axis. Cyan curves, no intercalator present. (d) Time traces recorded at 40 pN showing dsDNA digestion by the exonuclease activity of T7 DNA polymerase. Black, no intercalator; red, YO; blue, SxO; green, SbG. Under these experimental conditions, YOYO abolished all exonuclease activities. (e) Histograms of the resulting average digestion rates obtained at the four different conditions (Methods).

Mentions: Finally, we set out to investigate how intercalators and their kinetics have an impact on DNA overstretching and enzymatic processing of DNA. DNA overstretching is a cooperative transition during which the two complementary DNA strands can unwind and—depending on the salt conditions—separate (melt)35. It is characterized by a wide plateau in the force-extension curve at ∼65 pN (Fig. 7a). In the presence of the bis-intercalator YOYO, however, the force plateau was replaced by a monotonic force increase, demonstrating that the cooperativity of the transition was perturbed (Fig. 7a, red curve). The corresponding kymograph obtained from concomitantly measured fluorescence images showed that intercalator binding and DNA elongation occur homogeneously over the DNA molecule. These two observations suggest that intercalated DNA is stabilized and protected against unwinding and melting, and that overstretching cannot trigger intercalator unbinding. Similar results were obtained for the mono-intercalator SbG (Fig. 7b), indicating that these effects are not specific for bis-intercalators. Markedly different results were obtained in DNA-overstretching experiments with YO present in solution, which has a much faster dissociation rate than both YOYO and SbG: the kymograph in Fig. 7c shows that the binding pattern is relatively homogeneous at low extensions, converting to a heterogeneous pattern exhibiting extended ‘dark' regions at extensions above 18 μm. The force-extension curve, on the other hand, exhibits a relatively unperturbed force plateau. Both observations suggest that, despite YO being present in solution, the intercalated DNA could still overstretch in a cooperative manner and that the intercalator unbinds during the process of DNA unwinding and melting35. We investigated DNA intercalation under various conditions (Supplementary Fig. 5) and concluded that these differences in overstretching characteristics are explained by the differences in intercalator kinetics: an intercalator that exhibits an off-rate that is fast compared with the timescale of stretching (for example, YO in Fig. 7c) unbinds fast enough to not disturb the cooperative overstretching transition.


The impact of DNA intercalators on DNA and DNA-processing enzymes elucidated through force-dependent binding kinetics.

Biebricher AS, Heller I, Roijmans RF, Hoekstra TP, Peterman EJ, Wuite GJ - Nat Commun (2015)

Impact of intercalators on DNA overstretching and DNA-enzyme activity.(a–c) Kymographs and corresponding force-extension curves (red) in the presence of YOYO (a), SbG (b) and YO (c) at 100 mM NaCl. The kymograph image and corresponding force-extension curves are co-aligned along the horizontal axis, which is possible because of the constant stretching speed of the moving bead (visible as the bright, upward tilted bar in the image). Thus, the fluorescence pattern in the vertical direction in the kymograph is a DNA-staining pattern at the DNA extension indicated on the horizontal axis. Cyan curves, no intercalator present. (d) Time traces recorded at 40 pN showing dsDNA digestion by the exonuclease activity of T7 DNA polymerase. Black, no intercalator; red, YO; blue, SxO; green, SbG. Under these experimental conditions, YOYO abolished all exonuclease activities. (e) Histograms of the resulting average digestion rates obtained at the four different conditions (Methods).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4557362&req=5

f7: Impact of intercalators on DNA overstretching and DNA-enzyme activity.(a–c) Kymographs and corresponding force-extension curves (red) in the presence of YOYO (a), SbG (b) and YO (c) at 100 mM NaCl. The kymograph image and corresponding force-extension curves are co-aligned along the horizontal axis, which is possible because of the constant stretching speed of the moving bead (visible as the bright, upward tilted bar in the image). Thus, the fluorescence pattern in the vertical direction in the kymograph is a DNA-staining pattern at the DNA extension indicated on the horizontal axis. Cyan curves, no intercalator present. (d) Time traces recorded at 40 pN showing dsDNA digestion by the exonuclease activity of T7 DNA polymerase. Black, no intercalator; red, YO; blue, SxO; green, SbG. Under these experimental conditions, YOYO abolished all exonuclease activities. (e) Histograms of the resulting average digestion rates obtained at the four different conditions (Methods).
Mentions: Finally, we set out to investigate how intercalators and their kinetics have an impact on DNA overstretching and enzymatic processing of DNA. DNA overstretching is a cooperative transition during which the two complementary DNA strands can unwind and—depending on the salt conditions—separate (melt)35. It is characterized by a wide plateau in the force-extension curve at ∼65 pN (Fig. 7a). In the presence of the bis-intercalator YOYO, however, the force plateau was replaced by a monotonic force increase, demonstrating that the cooperativity of the transition was perturbed (Fig. 7a, red curve). The corresponding kymograph obtained from concomitantly measured fluorescence images showed that intercalator binding and DNA elongation occur homogeneously over the DNA molecule. These two observations suggest that intercalated DNA is stabilized and protected against unwinding and melting, and that overstretching cannot trigger intercalator unbinding. Similar results were obtained for the mono-intercalator SbG (Fig. 7b), indicating that these effects are not specific for bis-intercalators. Markedly different results were obtained in DNA-overstretching experiments with YO present in solution, which has a much faster dissociation rate than both YOYO and SbG: the kymograph in Fig. 7c shows that the binding pattern is relatively homogeneous at low extensions, converting to a heterogeneous pattern exhibiting extended ‘dark' regions at extensions above 18 μm. The force-extension curve, on the other hand, exhibits a relatively unperturbed force plateau. Both observations suggest that, despite YO being present in solution, the intercalated DNA could still overstretch in a cooperative manner and that the intercalator unbinds during the process of DNA unwinding and melting35. We investigated DNA intercalation under various conditions (Supplementary Fig. 5) and concluded that these differences in overstretching characteristics are explained by the differences in intercalator kinetics: an intercalator that exhibits an off-rate that is fast compared with the timescale of stretching (for example, YO in Fig. 7c) unbinds fast enough to not disturb the cooperative overstretching transition.

Bottom Line: Here we elucidate this perturbation by combining single-dye fluorescence microscopy with force spectroscopy and measuring the kinetics of DNA intercalation by the mono- and bis-intercalating cyanine dyes SYTOX Orange, SYTOX Green, SYBR Gold, YO-PRO-1, YOYO-1 and POPO-3.These rates can be tuned over a range of seven orders of magnitude by changing DNA tension, intercalating species and ionic strength.We show that optimizing these rates minimizes the impact of intercalators on strand separation and enzymatic activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics and Astronomy, LaserLaB Amsterdam, VU University Amsterdam, De Boelelaan 1081, Amsterdam 1081HV, The Netherlands.

ABSTRACT
DNA intercalators are widely used as fluorescent probes to visualize DNA and DNA transactions in vivo and in vitro. It is well known that they perturb DNA structure and stability, which can in turn influence DNA-processing by proteins. Here we elucidate this perturbation by combining single-dye fluorescence microscopy with force spectroscopy and measuring the kinetics of DNA intercalation by the mono- and bis-intercalating cyanine dyes SYTOX Orange, SYTOX Green, SYBR Gold, YO-PRO-1, YOYO-1 and POPO-3. We show that their DNA-binding affinity is mainly governed by a strongly tension-dependent dissociation rate. These rates can be tuned over a range of seven orders of magnitude by changing DNA tension, intercalating species and ionic strength. We show that optimizing these rates minimizes the impact of intercalators on strand separation and enzymatic activity. These new insights provide handles for the improved use of intercalators as DNA probes with minimal perturbation and maximal efficacy.

No MeSH data available.


Related in: MedlinePlus