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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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Single-molecule detection of cruciform extrusion. (A) Sketch of the experiment. A single DNA molecule containing an inverted repeat is tethered at one end to a glass surface and at the other end to a magnetic bead. A pair of magnets serves to control the vertical extending force, F, applied to the DNA and the number of rotations, n, imposed on the bead and hence the DNA supercoiling. The DNA end-to-end extension, l, is determined by measuring the position of the bead above the surface in real-time by computer-aided videomicroscopy. When the inverted repeat extrudes into a cruciform, unwinding of B-DNA titrates out negative supercoils and an increase, Δl, in DNA extension is observed. (B) Real-time data showing abrupt, reversible, changes in extension for Charomid 9-5 kb DNA (F ∼ 0.45 pN, n = −16 turns). Green points correspond to real-time data acquisition (60 Hz) while red points correspond to raw signal averaged over ∼1 s. Both structural (Δl) and kinetic (Twait and Tcruciform) features of the reaction can be obtained from these time-traces. (C) Histogram of transition amplitudes. The dashed line indicates the mean <Δl> = 241 ± 3 nm (n = 74 points). (D) Histograms of Twait (blue) and Tcruciform (red) and single-exponential fits to data. Mean lifetimes are <Twait> = 20.2 ± 1.2 s (n = 490 events) and <Tcruciform> = 13.5 ± 0.9 s (n = 489 events).
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Figure 1: Single-molecule detection of cruciform extrusion. (A) Sketch of the experiment. A single DNA molecule containing an inverted repeat is tethered at one end to a glass surface and at the other end to a magnetic bead. A pair of magnets serves to control the vertical extending force, F, applied to the DNA and the number of rotations, n, imposed on the bead and hence the DNA supercoiling. The DNA end-to-end extension, l, is determined by measuring the position of the bead above the surface in real-time by computer-aided videomicroscopy. When the inverted repeat extrudes into a cruciform, unwinding of B-DNA titrates out negative supercoils and an increase, Δl, in DNA extension is observed. (B) Real-time data showing abrupt, reversible, changes in extension for Charomid 9-5 kb DNA (F ∼ 0.45 pN, n = −16 turns). Green points correspond to real-time data acquisition (60 Hz) while red points correspond to raw signal averaged over ∼1 s. Both structural (Δl) and kinetic (Twait and Tcruciform) features of the reaction can be obtained from these time-traces. (C) Histogram of transition amplitudes. The dashed line indicates the mean <Δl> = 241 ± 3 nm (n = 74 points). (D) Histograms of Twait (blue) and Tcruciform (red) and single-exponential fits to data. Mean lifetimes are <Twait> = 20.2 ± 1.2 s (n = 490 events) and <Tcruciform> = 13.5 ± 0.9 s (n = 489 events).

Mentions: A cruciform is a structure based on a DNA inverted repeat, and characterized by the presence of a four-way junction in which two of the branches are hairpin structures formed on each strand of the inverted repeat (Figure 1A). The bases located between the inverted repeats do not self-pair and instead form the apical loops of the hairpins. Although the four-way junction and the apical loops render the cruciform less stable than B-form DNA, negative DNA supercoiling has long been known to stabilize cruciform structures (1–4).Figure 1.


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

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

Single-molecule detection of cruciform extrusion. (A) Sketch of the experiment. A single DNA molecule containing an inverted repeat is tethered at one end to a glass surface and at the other end to a magnetic bead. A pair of magnets serves to control the vertical extending force, F, applied to the DNA and the number of rotations, n, imposed on the bead and hence the DNA supercoiling. The DNA end-to-end extension, l, is determined by measuring the position of the bead above the surface in real-time by computer-aided videomicroscopy. When the inverted repeat extrudes into a cruciform, unwinding of B-DNA titrates out negative supercoils and an increase, Δl, in DNA extension is observed. (B) Real-time data showing abrupt, reversible, changes in extension for Charomid 9-5 kb DNA (F ∼ 0.45 pN, n = −16 turns). Green points correspond to real-time data acquisition (60 Hz) while red points correspond to raw signal averaged over ∼1 s. Both structural (Δl) and kinetic (Twait and Tcruciform) features of the reaction can be obtained from these time-traces. (C) Histogram of transition amplitudes. The dashed line indicates the mean <Δl> = 241 ± 3 nm (n = 74 points). (D) Histograms of Twait (blue) and Tcruciform (red) and single-exponential fits to data. Mean lifetimes are <Twait> = 20.2 ± 1.2 s (n = 490 events) and <Tcruciform> = 13.5 ± 0.9 s (n = 489 events).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Single-molecule detection of cruciform extrusion. (A) Sketch of the experiment. A single DNA molecule containing an inverted repeat is tethered at one end to a glass surface and at the other end to a magnetic bead. A pair of magnets serves to control the vertical extending force, F, applied to the DNA and the number of rotations, n, imposed on the bead and hence the DNA supercoiling. The DNA end-to-end extension, l, is determined by measuring the position of the bead above the surface in real-time by computer-aided videomicroscopy. When the inverted repeat extrudes into a cruciform, unwinding of B-DNA titrates out negative supercoils and an increase, Δl, in DNA extension is observed. (B) Real-time data showing abrupt, reversible, changes in extension for Charomid 9-5 kb DNA (F ∼ 0.45 pN, n = −16 turns). Green points correspond to real-time data acquisition (60 Hz) while red points correspond to raw signal averaged over ∼1 s. Both structural (Δl) and kinetic (Twait and Tcruciform) features of the reaction can be obtained from these time-traces. (C) Histogram of transition amplitudes. The dashed line indicates the mean <Δl> = 241 ± 3 nm (n = 74 points). (D) Histograms of Twait (blue) and Tcruciform (red) and single-exponential fits to data. Mean lifetimes are <Twait> = 20.2 ± 1.2 s (n = 490 events) and <Tcruciform> = 13.5 ± 0.9 s (n = 489 events).
Mentions: A cruciform is a structure based on a DNA inverted repeat, and characterized by the presence of a four-way junction in which two of the branches are hairpin structures formed on each strand of the inverted repeat (Figure 1A). The bases located between the inverted repeats do not self-pair and instead form the apical loops of the hairpins. Although the four-way junction and the apical loops render the cruciform less stable than B-form DNA, negative DNA supercoiling has long been known to stabilize cruciform structures (1–4).Figure 1.

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