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Real-time detection of cruciform extrusion by single-molecule DNA nanomanipulation.

Ramreddy T, Sachidanandam R, Strick TR - Nucleic Acids Res. (2011)

Bottom Line: Using mutational analysis and a simple two-state model, we find that in the transition state intermediate only the B-DNA located between the inverted repeats (and corresponding to the unpaired apical loop) is unwound, implying that initial stabilization of the four-way (or Holliday) junction is rate-limiting.We thus find that cruciform extrusion is kinetically regulated by features of the hairpin loop, while rewinding is kinetically regulated by features of the stem.These results provide mechanistic insight into cruciform extrusion and help understand the structural features that determine the relative stability of the cruciform and B-form states.

View Article: PubMed Central - PubMed

Affiliation: Institut Jacques Monod, CNRS UMR 7592, University of Paris - Diderot, 15 rue Hélène Brion, 75205 Paris Cedex 13, France.

ABSTRACT
During cruciform extrusion, a DNA inverted repeat unwinds and forms a four-way junction in which two of the branches consist of hairpin structures obtained by self-pairing of the inverted repeats. Here, we use single-molecule DNA nanomanipulation to monitor in real-time cruciform extrusion and rewinding. This allows us to determine the size of the cruciform to nearly base pair accuracy and its kinetics with second-scale time resolution. We present data obtained with two different inverted repeats, one perfect and one imperfect, and extend single-molecule force spectroscopy to measure the torque dependence of cruciform extrusion and rewinding kinetics. Using mutational analysis and a simple two-state model, we find that in the transition state intermediate only the B-DNA located between the inverted repeats (and corresponding to the unpaired apical loop) is unwound, implying that initial stabilization of the four-way (or Holliday) junction is rate-limiting. We thus find that cruciform extrusion is kinetically regulated by features of the hairpin loop, while rewinding is kinetically regulated by features of the stem. These results provide mechanistic insight into cruciform extrusion and help understand the structural features that determine the relative stability of the cruciform and B-form states.

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Kinetic analysis of cruciform extrusion and rewinding for Charomid 9-5 kb DNA. (A) As negative DNA supercoiling increases, progressively less time is spent in the B-DNA state and more time is spent in the extruded-cruciform state. Experiments were performed at F = 0.45 pN. (B) Free energy barrier to cruciform extrusion (blue) and rewinding (red) as a function of negative supercoiling. The barrier to cruciform extrusion decreases linearly with negative supercoiling at a rate of 0.31 ± 0.015 kBT/turn (B-DNA is destabilized by unwinding), while the barrier to rewinding increases linearly by 0.71 ± 0.014 kBT/turn (cruciform DNA is stabilized by unwinding). At least ∼300 events were measured at each rotation point to determine each mean transition rate.
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Figure 3: Kinetic analysis of cruciform extrusion and rewinding for Charomid 9-5 kb DNA. (A) As negative DNA supercoiling increases, progressively less time is spent in the B-DNA state and more time is spent in the extruded-cruciform state. Experiments were performed at F = 0.45 pN. (B) Free energy barrier to cruciform extrusion (blue) and rewinding (red) as a function of negative supercoiling. The barrier to cruciform extrusion decreases linearly with negative supercoiling at a rate of 0.31 ± 0.015 kBT/turn (B-DNA is destabilized by unwinding), while the barrier to rewinding increases linearly by 0.71 ± 0.014 kBT/turn (cruciform DNA is stabilized by unwinding). At least ∼300 events were measured at each rotation point to determine each mean transition rate.

Mentions: The time elapsed before an extrusion event, Twait, and the lifetime of the extruded state, Tcruciform, can be directly determined from the time-traces (Figure 1B). Histograms of Twait and Tcruciform display single-exponential lifetime distributions (Figure 1D), suggesting that at the temporal resolution of the experiment (∼1 s) a single energy barrier dominates the transition between the B-DNA and cruciform states. These measurements were then repeated on DNA for different levels of negative supercoiling (Figure 3A). The data show that as the DNA is progressively unwound, the cruciform state becomes more likely as a result of an increase in the rate of cruciform extrusion, kf = 1/<Twait>, and a concomitant decrease in the rate of cruciform rewinding, kr = 1/<Tcruciform>. More precisely, kf increases exponentially while kr decreases exponentially with negative supercoiling as per an Arrhenius law (Figure 3B). At the same time, we find that ncruciform remains essentially constant, indicating that the size of the cruciform does not increase with negative supercoiling (Supplementary Figure S3). These results imply that the cruciform is fully formed upon extrusion, and that the hairpin branches cannot be extended beyond the inverted repeats. Instead, the cruciform becomes progressively more stable with negative supercoiling, consistent with mechanical stabilization of the extruded cruciform by the torque generated by negative supercoiling.Figure 3.


Real-time detection of cruciform extrusion by single-molecule DNA nanomanipulation.

Ramreddy T, Sachidanandam R, Strick TR - Nucleic Acids Res. (2011)

Kinetic analysis of cruciform extrusion and rewinding for Charomid 9-5 kb DNA. (A) As negative DNA supercoiling increases, progressively less time is spent in the B-DNA state and more time is spent in the extruded-cruciform state. Experiments were performed at F = 0.45 pN. (B) Free energy barrier to cruciform extrusion (blue) and rewinding (red) as a function of negative supercoiling. The barrier to cruciform extrusion decreases linearly with negative supercoiling at a rate of 0.31 ± 0.015 kBT/turn (B-DNA is destabilized by unwinding), while the barrier to rewinding increases linearly by 0.71 ± 0.014 kBT/turn (cruciform DNA is stabilized by unwinding). At least ∼300 events were measured at each rotation point to determine each mean transition rate.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105387&req=5

Figure 3: Kinetic analysis of cruciform extrusion and rewinding for Charomid 9-5 kb DNA. (A) As negative DNA supercoiling increases, progressively less time is spent in the B-DNA state and more time is spent in the extruded-cruciform state. Experiments were performed at F = 0.45 pN. (B) Free energy barrier to cruciform extrusion (blue) and rewinding (red) as a function of negative supercoiling. The barrier to cruciform extrusion decreases linearly with negative supercoiling at a rate of 0.31 ± 0.015 kBT/turn (B-DNA is destabilized by unwinding), while the barrier to rewinding increases linearly by 0.71 ± 0.014 kBT/turn (cruciform DNA is stabilized by unwinding). At least ∼300 events were measured at each rotation point to determine each mean transition rate.
Mentions: The time elapsed before an extrusion event, Twait, and the lifetime of the extruded state, Tcruciform, can be directly determined from the time-traces (Figure 1B). Histograms of Twait and Tcruciform display single-exponential lifetime distributions (Figure 1D), suggesting that at the temporal resolution of the experiment (∼1 s) a single energy barrier dominates the transition between the B-DNA and cruciform states. These measurements were then repeated on DNA for different levels of negative supercoiling (Figure 3A). The data show that as the DNA is progressively unwound, the cruciform state becomes more likely as a result of an increase in the rate of cruciform extrusion, kf = 1/<Twait>, and a concomitant decrease in the rate of cruciform rewinding, kr = 1/<Tcruciform>. More precisely, kf increases exponentially while kr decreases exponentially with negative supercoiling as per an Arrhenius law (Figure 3B). At the same time, we find that ncruciform remains essentially constant, indicating that the size of the cruciform does not increase with negative supercoiling (Supplementary Figure S3). These results imply that the cruciform is fully formed upon extrusion, and that the hairpin branches cannot be extended beyond the inverted repeats. Instead, the cruciform becomes progressively more stable with negative supercoiling, consistent with mechanical stabilization of the extruded cruciform by the torque generated by negative supercoiling.Figure 3.

Bottom Line: Using mutational analysis and a simple two-state model, we find that in the transition state intermediate only the B-DNA located between the inverted repeats (and corresponding to the unpaired apical loop) is unwound, implying that initial stabilization of the four-way (or Holliday) junction is rate-limiting.We thus find that cruciform extrusion is kinetically regulated by features of the hairpin loop, while rewinding is kinetically regulated by features of the stem.These results provide mechanistic insight into cruciform extrusion and help understand the structural features that determine the relative stability of the cruciform and B-form states.

View Article: PubMed Central - PubMed

Affiliation: Institut Jacques Monod, CNRS UMR 7592, University of Paris - Diderot, 15 rue Hélène Brion, 75205 Paris Cedex 13, France.

ABSTRACT
During cruciform extrusion, a DNA inverted repeat unwinds and forms a four-way junction in which two of the branches consist of hairpin structures obtained by self-pairing of the inverted repeats. Here, we use single-molecule DNA nanomanipulation to monitor in real-time cruciform extrusion and rewinding. This allows us to determine the size of the cruciform to nearly base pair accuracy and its kinetics with second-scale time resolution. We present data obtained with two different inverted repeats, one perfect and one imperfect, and extend single-molecule force spectroscopy to measure the torque dependence of cruciform extrusion and rewinding kinetics. Using mutational analysis and a simple two-state model, we find that in the transition state intermediate only the B-DNA located between the inverted repeats (and corresponding to the unpaired apical loop) is unwound, implying that initial stabilization of the four-way (or Holliday) junction is rate-limiting. We thus find that cruciform extrusion is kinetically regulated by features of the hairpin loop, while rewinding is kinetically regulated by features of the stem. These results provide mechanistic insight into cruciform extrusion and help understand the structural features that determine the relative stability of the cruciform and B-form states.

Show MeSH
Related in: MedlinePlus