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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 and structural analysis of cruciform extrusion in mutColE1 inverted repeats bearing (A–C) a 5 base loop or (D–F) an 8 base loop. (A) The mutColE1-5b inverted repeat is seen (B) to extrude reversibly as shown previously in Figure 4. (C) The free energy barrier to cruciform rewinding (red) is more sensitive to torque than the free energy barrier to cruciform extrusion (blue), d ln kf/d ln kr = 4.2 ± 0.6. (D) The mutColE1-8b inverted repeat with an 8 base loop is seen (E) to extrude with slower kinetics than the 5-base loop. (F) The free energy barrier to rewinding is still more sensitive to torque than the free energy barrier to extrusion, but less so: d ln kf/d ln kr = 2.6 ± 0.5. (G) Two-state model for torque-induced cruciform extrusion. The transition-state intermediate to cruciform extrusion is formed by unwinding B-DNA by nf turns, and for complete formation of the cruciform the B-DNA must thereafter be unwound by an additional nr turns, with nf + nr = ncruciform. The relative sensitivity of transition rates kf and kr to supercoiling predicts the position of the transition state between the two states, d ln kf/d ln kr = nf/nr.
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Figure 5: Kinetic and structural analysis of cruciform extrusion in mutColE1 inverted repeats bearing (A–C) a 5 base loop or (D–F) an 8 base loop. (A) The mutColE1-5b inverted repeat is seen (B) to extrude reversibly as shown previously in Figure 4. (C) The free energy barrier to cruciform rewinding (red) is more sensitive to torque than the free energy barrier to cruciform extrusion (blue), d ln kf/d ln kr = 4.2 ± 0.6. (D) The mutColE1-8b inverted repeat with an 8 base loop is seen (E) to extrude with slower kinetics than the 5-base loop. (F) The free energy barrier to rewinding is still more sensitive to torque than the free energy barrier to extrusion, but less so: d ln kf/d ln kr = 2.6 ± 0.5. (G) Two-state model for torque-induced cruciform extrusion. The transition-state intermediate to cruciform extrusion is formed by unwinding B-DNA by nf turns, and for complete formation of the cruciform the B-DNA must thereafter be unwound by an additional nr turns, with nf + nr = ncruciform. The relative sensitivity of transition rates kf and kr to supercoiling predicts the position of the transition state between the two states, d ln kf/d ln kr = nf/nr.

Mentions: As can be done with force spectroscopy (37,38), the supercoiling dependence of kf and kr can be quantitatively analyzed to determine the ratio nr/nf, where nf is the amount of DNA unwinding separating the B-DNA state from the transition state, nr is the amount of DNA unwinding required to complete extrusion, i.e. to go from the transition state to the cruciform state and nf + nr = ncruciform is well-determined experimentally (for details, see Supplementary Data and Figure 5E). We thus find for the Charomid X quasipalindrome nr/nf = 2.3 ± 0.1. Using nr + nf = ncruciform = 34 ± 1 bp, we interpret this as meaning that in the transition state 10 ± 1 bp of DNA are unwound. Note that the Charomid X cruciform is predicted to have a large, weakly structured and presumably degenerate ∼12 base loop region at its apex (Figure 2C). [We further note that this result is independent of the extending force used during the measurement, as in the range of forces tested increasing the force leaves the relative position of the transition state essentially unchanged (Supplementary Figure S4).] Based on the simple two-state model the data permit for, these experiments suggest that the transition-state intermediate to cruciform extrusion corresponds to a state where the DNA is unwound only in the apical loop region of the cruciform.


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

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

Kinetic and structural analysis of cruciform extrusion in mutColE1 inverted repeats bearing (A–C) a 5 base loop or (D–F) an 8 base loop. (A) The mutColE1-5b inverted repeat is seen (B) to extrude reversibly as shown previously in Figure 4. (C) The free energy barrier to cruciform rewinding (red) is more sensitive to torque than the free energy barrier to cruciform extrusion (blue), d ln kf/d ln kr = 4.2 ± 0.6. (D) The mutColE1-8b inverted repeat with an 8 base loop is seen (E) to extrude with slower kinetics than the 5-base loop. (F) The free energy barrier to rewinding is still more sensitive to torque than the free energy barrier to extrusion, but less so: d ln kf/d ln kr = 2.6 ± 0.5. (G) Two-state model for torque-induced cruciform extrusion. The transition-state intermediate to cruciform extrusion is formed by unwinding B-DNA by nf turns, and for complete formation of the cruciform the B-DNA must thereafter be unwound by an additional nr turns, with nf + nr = ncruciform. The relative sensitivity of transition rates kf and kr to supercoiling predicts the position of the transition state between the two states, d ln kf/d ln kr = nf/nr.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: Kinetic and structural analysis of cruciform extrusion in mutColE1 inverted repeats bearing (A–C) a 5 base loop or (D–F) an 8 base loop. (A) The mutColE1-5b inverted repeat is seen (B) to extrude reversibly as shown previously in Figure 4. (C) The free energy barrier to cruciform rewinding (red) is more sensitive to torque than the free energy barrier to cruciform extrusion (blue), d ln kf/d ln kr = 4.2 ± 0.6. (D) The mutColE1-8b inverted repeat with an 8 base loop is seen (E) to extrude with slower kinetics than the 5-base loop. (F) The free energy barrier to rewinding is still more sensitive to torque than the free energy barrier to extrusion, but less so: d ln kf/d ln kr = 2.6 ± 0.5. (G) Two-state model for torque-induced cruciform extrusion. The transition-state intermediate to cruciform extrusion is formed by unwinding B-DNA by nf turns, and for complete formation of the cruciform the B-DNA must thereafter be unwound by an additional nr turns, with nf + nr = ncruciform. The relative sensitivity of transition rates kf and kr to supercoiling predicts the position of the transition state between the two states, d ln kf/d ln kr = nf/nr.
Mentions: As can be done with force spectroscopy (37,38), the supercoiling dependence of kf and kr can be quantitatively analyzed to determine the ratio nr/nf, where nf is the amount of DNA unwinding separating the B-DNA state from the transition state, nr is the amount of DNA unwinding required to complete extrusion, i.e. to go from the transition state to the cruciform state and nf + nr = ncruciform is well-determined experimentally (for details, see Supplementary Data and Figure 5E). We thus find for the Charomid X quasipalindrome nr/nf = 2.3 ± 0.1. Using nr + nf = ncruciform = 34 ± 1 bp, we interpret this as meaning that in the transition state 10 ± 1 bp of DNA are unwound. Note that the Charomid X cruciform is predicted to have a large, weakly structured and presumably degenerate ∼12 base loop region at its apex (Figure 2C). [We further note that this result is independent of the extending force used during the measurement, as in the range of forces tested increasing the force leaves the relative position of the transition state essentially unchanged (Supplementary Figure S4).] Based on the simple two-state model the data permit for, these experiments suggest that the transition-state intermediate to cruciform extrusion corresponds to a state where the DNA is unwound only in the apical loop region of the cruciform.

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