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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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Irreversible and reversible cruciform extrusion on the wtColE1 inverted repeat and its derivative mutColE1-5b. (A) The wtColE1 inverted repeat forms a perfect cruciform (B) in an irreversible fashion under conditions of negative supercoiling (n = −21). Immediately after the extrusion event, supercoiling is returned to n = −19 rotations to favor rewinding. Despite this no complete rewinding is observed, although unsuccessful attempts at refolding are regularly observed (down arrows). (C) The mutColE1-5b inverted repeat extrudes into an imperfect cruciform (D) in a reversible fashion, allowing for statistical analysis of the kinetics of cruciform extrusion and rewinding. The increased level of noise in the extension of the B-DNA state is due to the presence of A + T-rich regions flanking the inverted repeat (see Supplementary Figure S7).
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Figure 4: Irreversible and reversible cruciform extrusion on the wtColE1 inverted repeat and its derivative mutColE1-5b. (A) The wtColE1 inverted repeat forms a perfect cruciform (B) in an irreversible fashion under conditions of negative supercoiling (n = −21). Immediately after the extrusion event, supercoiling is returned to n = −19 rotations to favor rewinding. Despite this no complete rewinding is observed, although unsuccessful attempts at refolding are regularly observed (down arrows). (C) The mutColE1-5b inverted repeat extrudes into an imperfect cruciform (D) in a reversible fashion, allowing for statistical analysis of the kinetics of cruciform extrusion and rewinding. The increased level of noise in the extension of the B-DNA state is due to the presence of A + T-rich regions flanking the inverted repeat (see Supplementary Figure S7).

Mentions: To further test our model, we performed another series of experiments investigating the kinetics of extrusion and rewinding for the canonical 31 bp ColE1 perfect inverted repeat, a cruciform characterized by a 5-base apical loop and perfectly paired 13 bp stem (Figure 4A) (1). We selected this sequence based on its perfect pairing and its reported propensity for irreversible extrusion, as it could potentially provide a basis on which to test modifications leading to reversible cruciform extrusion. Single-molecule analysis of the wtColE1 inverted repeat subjected to negative supercoiling indeed showed that cruciform extrusion was essentially irreversible, with a large increase in DNA extension during cruciform extrusion which never fully reversed. Nevertheless, the data indicate that the extruded cruciform can make attempts at refolding (Figure 4B). During such attempts, the extension of the system can nearly return to that of the native low-extension state. However, these attempts are unsuccessful and do not correspond to full rewinding of the cruciform, as their lifetime, on the order of seconds, is much shorter than the lifetime of the true B-DNA state (410 ± 100 s, see Supplementary Figure S5). In the context of our two-state model describing the relative position of the transition state, these results suggest that the transition state to rewinding lies very close to the native state for this perfect inverted repeat. Further experiments (Supplementary Figure S6) show that rewinding of the cruciform can occur when the DNA is returned to n = −9 turns but that this is still a slow process. Thus under the conditions in which this perfect inverted repeat extrudes, it is thereafter kinetically trapped and thus not a good thermodynamic system.Figure 4.


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

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

Irreversible and reversible cruciform extrusion on the wtColE1 inverted repeat and its derivative mutColE1-5b. (A) The wtColE1 inverted repeat forms a perfect cruciform (B) in an irreversible fashion under conditions of negative supercoiling (n = −21). Immediately after the extrusion event, supercoiling is returned to n = −19 rotations to favor rewinding. Despite this no complete rewinding is observed, although unsuccessful attempts at refolding are regularly observed (down arrows). (C) The mutColE1-5b inverted repeat extrudes into an imperfect cruciform (D) in a reversible fashion, allowing for statistical analysis of the kinetics of cruciform extrusion and rewinding. The increased level of noise in the extension of the B-DNA state is due to the presence of A + T-rich regions flanking the inverted repeat (see Supplementary Figure S7).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Irreversible and reversible cruciform extrusion on the wtColE1 inverted repeat and its derivative mutColE1-5b. (A) The wtColE1 inverted repeat forms a perfect cruciform (B) in an irreversible fashion under conditions of negative supercoiling (n = −21). Immediately after the extrusion event, supercoiling is returned to n = −19 rotations to favor rewinding. Despite this no complete rewinding is observed, although unsuccessful attempts at refolding are regularly observed (down arrows). (C) The mutColE1-5b inverted repeat extrudes into an imperfect cruciform (D) in a reversible fashion, allowing for statistical analysis of the kinetics of cruciform extrusion and rewinding. The increased level of noise in the extension of the B-DNA state is due to the presence of A + T-rich regions flanking the inverted repeat (see Supplementary Figure S7).
Mentions: To further test our model, we performed another series of experiments investigating the kinetics of extrusion and rewinding for the canonical 31 bp ColE1 perfect inverted repeat, a cruciform characterized by a 5-base apical loop and perfectly paired 13 bp stem (Figure 4A) (1). We selected this sequence based on its perfect pairing and its reported propensity for irreversible extrusion, as it could potentially provide a basis on which to test modifications leading to reversible cruciform extrusion. Single-molecule analysis of the wtColE1 inverted repeat subjected to negative supercoiling indeed showed that cruciform extrusion was essentially irreversible, with a large increase in DNA extension during cruciform extrusion which never fully reversed. Nevertheless, the data indicate that the extruded cruciform can make attempts at refolding (Figure 4B). During such attempts, the extension of the system can nearly return to that of the native low-extension state. However, these attempts are unsuccessful and do not correspond to full rewinding of the cruciform, as their lifetime, on the order of seconds, is much shorter than the lifetime of the true B-DNA state (410 ± 100 s, see Supplementary Figure S5). In the context of our two-state model describing the relative position of the transition state, these results suggest that the transition state to rewinding lies very close to the native state for this perfect inverted repeat. Further experiments (Supplementary Figure S6) show that rewinding of the cruciform can occur when the DNA is returned to n = −9 turns but that this is still a slow process. Thus under the conditions in which this perfect inverted repeat extrudes, it is thereafter kinetically trapped and thus not a good thermodynamic system.Figure 4.

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