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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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Related in: MedlinePlus

Single-molecule screen of Charomid 9-5 kb is used to identify the Charomid X quasipalindrome. (A) Screen strategy. Charomid 9-5 kb is derived from Charomid 9-11 kb by removing the 6 kb region of the plasmid, corresponding to three consecutive repeats of a 2-kb fragment (35). Then, five constructs are derived from Charomid 9-5 kb using the PCR reaction, each construct lacking a different 1 kb segment of the plasmid. (B) Time-traces for the five constructs described above: first level of screening. DNA constructs were unwound by n = −12 turns using the magnetic trap, and their extension monitored in real-time. The Charomid 9-5 kb displays the same behavior as the Charomid 9-11 kb plasmid, indicating that the repeat region is not responsible for the observed fluctuations. The Charo-S3 construct is the only one of the five constructs to lack the canonical fluctuations observed on the original plasmid, indicating that the sequence responsible for this behavior is located in the corresponding 1 kb of DNA, which is specifically lacking in the construct. Fast residual fluctuations indicate denaturation or cruciform extrusion on secondary sequences in the plasmid (C) Screen results. The above process was reiterated two times to narrow down the sequence responsible for the observed fluctuations. The 33-bp Charomid X sequence responsible for the observed fluctuations is a quasipalindrome, which extrudes to form a complex and unstable cruciform. A putative minimum energy fold derived from mfold for high-salt conditions (0.5 M NaCl) is presented; in the lower salt conditions initially used here, the 3-bp separating the two apical loops are unlikely to be stable at room temperature, implying that the apical loop is in fact the sum of the two smaller loops. This is consistent with the extensive amount of unwinding estimated to be present in the transition state. The mutation made to increase the loop size involved converting the G circled in red into a T, disrupting a crucial base pair for high-salt conditions but not low-salt conditions (Table 1).
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Figure 2: Single-molecule screen of Charomid 9-5 kb is used to identify the Charomid X quasipalindrome. (A) Screen strategy. Charomid 9-5 kb is derived from Charomid 9-11 kb by removing the 6 kb region of the plasmid, corresponding to three consecutive repeats of a 2-kb fragment (35). Then, five constructs are derived from Charomid 9-5 kb using the PCR reaction, each construct lacking a different 1 kb segment of the plasmid. (B) Time-traces for the five constructs described above: first level of screening. DNA constructs were unwound by n = −12 turns using the magnetic trap, and their extension monitored in real-time. The Charomid 9-5 kb displays the same behavior as the Charomid 9-11 kb plasmid, indicating that the repeat region is not responsible for the observed fluctuations. The Charo-S3 construct is the only one of the five constructs to lack the canonical fluctuations observed on the original plasmid, indicating that the sequence responsible for this behavior is located in the corresponding 1 kb of DNA, which is specifically lacking in the construct. Fast residual fluctuations indicate denaturation or cruciform extrusion on secondary sequences in the plasmid (C) Screen results. The above process was reiterated two times to narrow down the sequence responsible for the observed fluctuations. The 33-bp Charomid X sequence responsible for the observed fluctuations is a quasipalindrome, which extrudes to form a complex and unstable cruciform. A putative minimum energy fold derived from mfold for high-salt conditions (0.5 M NaCl) is presented; in the lower salt conditions initially used here, the 3-bp separating the two apical loops are unlikely to be stable at room temperature, implying that the apical loop is in fact the sum of the two smaller loops. This is consistent with the extensive amount of unwinding estimated to be present in the transition state. The mutation made to increase the loop size involved converting the G circled in red into a T, disrupting a crucial base pair for high-salt conditions but not low-salt conditions (Table 1).

Mentions: In Figure 1B, we present a real-time measurement of the change in DNA extension resulting from reversible extrusion of the Charomid X imperfect inverted repeat under conditions of negative supercoiling. The time-trace shows the 5-kb DNA undergoing abrupt transitions (‘hopping’) between a low-extension state and a high-extension state. The Charomid X sequence responsible for these fluctuations was identified by selectively removing different regions of the Charomid 9-5 kb DNA and screening the resulting constructs for the slow, large-scale fluctuations (Figure 2). The low- and high-extension states we observe are separated by a remarkably large change in DNA extension of <Δl> = 241 ± 3 nm (Figure 1C), nearly 15% of the full contour length of the DNA. No such slow, large-scale fluctuations are observed if the Charomid X sequence is positively supercoiled (Supplementary Figure S1). We thus estimate that when the DNA hops from the low-extension state to the high-extension state, <Δl>/δ = 3.3 units of writhe are titrated out, consistent with formation of a cruciform involving Ncruciform = 34 bp. This is in agreement with the expected size of the cruciform based on the Charomid X quasipalindromic sequence (33 bp, see Figure 2C). The Charomid X sequence still undergoes these fluctuations if separated from its original flanking sequences (Supplementary Figure S2).Figure 2.


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

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

Single-molecule screen of Charomid 9-5 kb is used to identify the Charomid X quasipalindrome. (A) Screen strategy. Charomid 9-5 kb is derived from Charomid 9-11 kb by removing the 6 kb region of the plasmid, corresponding to three consecutive repeats of a 2-kb fragment (35). Then, five constructs are derived from Charomid 9-5 kb using the PCR reaction, each construct lacking a different 1 kb segment of the plasmid. (B) Time-traces for the five constructs described above: first level of screening. DNA constructs were unwound by n = −12 turns using the magnetic trap, and their extension monitored in real-time. The Charomid 9-5 kb displays the same behavior as the Charomid 9-11 kb plasmid, indicating that the repeat region is not responsible for the observed fluctuations. The Charo-S3 construct is the only one of the five constructs to lack the canonical fluctuations observed on the original plasmid, indicating that the sequence responsible for this behavior is located in the corresponding 1 kb of DNA, which is specifically lacking in the construct. Fast residual fluctuations indicate denaturation or cruciform extrusion on secondary sequences in the plasmid (C) Screen results. The above process was reiterated two times to narrow down the sequence responsible for the observed fluctuations. The 33-bp Charomid X sequence responsible for the observed fluctuations is a quasipalindrome, which extrudes to form a complex and unstable cruciform. A putative minimum energy fold derived from mfold for high-salt conditions (0.5 M NaCl) is presented; in the lower salt conditions initially used here, the 3-bp separating the two apical loops are unlikely to be stable at room temperature, implying that the apical loop is in fact the sum of the two smaller loops. This is consistent with the extensive amount of unwinding estimated to be present in the transition state. The mutation made to increase the loop size involved converting the G circled in red into a T, disrupting a crucial base pair for high-salt conditions but not low-salt conditions (Table 1).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: Single-molecule screen of Charomid 9-5 kb is used to identify the Charomid X quasipalindrome. (A) Screen strategy. Charomid 9-5 kb is derived from Charomid 9-11 kb by removing the 6 kb region of the plasmid, corresponding to three consecutive repeats of a 2-kb fragment (35). Then, five constructs are derived from Charomid 9-5 kb using the PCR reaction, each construct lacking a different 1 kb segment of the plasmid. (B) Time-traces for the five constructs described above: first level of screening. DNA constructs were unwound by n = −12 turns using the magnetic trap, and their extension monitored in real-time. The Charomid 9-5 kb displays the same behavior as the Charomid 9-11 kb plasmid, indicating that the repeat region is not responsible for the observed fluctuations. The Charo-S3 construct is the only one of the five constructs to lack the canonical fluctuations observed on the original plasmid, indicating that the sequence responsible for this behavior is located in the corresponding 1 kb of DNA, which is specifically lacking in the construct. Fast residual fluctuations indicate denaturation or cruciform extrusion on secondary sequences in the plasmid (C) Screen results. The above process was reiterated two times to narrow down the sequence responsible for the observed fluctuations. The 33-bp Charomid X sequence responsible for the observed fluctuations is a quasipalindrome, which extrudes to form a complex and unstable cruciform. A putative minimum energy fold derived from mfold for high-salt conditions (0.5 M NaCl) is presented; in the lower salt conditions initially used here, the 3-bp separating the two apical loops are unlikely to be stable at room temperature, implying that the apical loop is in fact the sum of the two smaller loops. This is consistent with the extensive amount of unwinding estimated to be present in the transition state. The mutation made to increase the loop size involved converting the G circled in red into a T, disrupting a crucial base pair for high-salt conditions but not low-salt conditions (Table 1).
Mentions: In Figure 1B, we present a real-time measurement of the change in DNA extension resulting from reversible extrusion of the Charomid X imperfect inverted repeat under conditions of negative supercoiling. The time-trace shows the 5-kb DNA undergoing abrupt transitions (‘hopping’) between a low-extension state and a high-extension state. The Charomid X sequence responsible for these fluctuations was identified by selectively removing different regions of the Charomid 9-5 kb DNA and screening the resulting constructs for the slow, large-scale fluctuations (Figure 2). The low- and high-extension states we observe are separated by a remarkably large change in DNA extension of <Δl> = 241 ± 3 nm (Figure 1C), nearly 15% of the full contour length of the DNA. No such slow, large-scale fluctuations are observed if the Charomid X sequence is positively supercoiled (Supplementary Figure S1). We thus estimate that when the DNA hops from the low-extension state to the high-extension state, <Δl>/δ = 3.3 units of writhe are titrated out, consistent with formation of a cruciform involving Ncruciform = 34 bp. This is in agreement with the expected size of the cruciform based on the Charomid X quasipalindromic sequence (33 bp, see Figure 2C). The Charomid X sequence still undergoes these fluctuations if separated from its original flanking sequences (Supplementary Figure S2).Figure 2.

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