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The flexibility of locally melted DNA.

Forties RA, Bundschuh R, Poirier MG - Nucleic Acids Res. (2009)

Bottom Line: One proposed explanation suggests that local melting of a few base pairs introduces flexible hinges.We have expanded this model to incorporate sequence and temperature dependence of the local melting, and tested it for three sequences at temperatures from 23 degrees C to 42 degrees C.We find that small melted bubbles are significantly more flexible than double-stranded DNA and can alter DNA flexibility at physiological temperatures.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, The Ohio State University, 191 West Woodruff Avenue, Columbus, OH 43210-1117, USA.

ABSTRACT
Protein-bound duplex DNA is often bent or kinked. Yet, quantification of intrinsic DNA bending that might lead to such protein interactions remains enigmatic. DNA cyclization experiments have indicated that DNA may form sharp bends more easily than predicted by the established worm-like chain (WLC) model. One proposed explanation suggests that local melting of a few base pairs introduces flexible hinges. We have expanded this model to incorporate sequence and temperature dependence of the local melting, and tested it for three sequences at temperatures from 23 degrees C to 42 degrees C. We find that small melted bubbles are significantly more flexible than double-stranded DNA and can alter DNA flexibility at physiological temperatures. However, these bubbles are not flexible enough to explain the recently observed very sharp bends in DNA.

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Related in: MedlinePlus

Example gel showing a ligation time course for the 116o sequence at a concentration of 0.33 nM ligated at 37°C with 50 U/ml T4 ligase. (a) Gel image showing both linearly and circularly ligated products. The bands in the gel are (from bottom to top) linear monomer (LM), linear dimer (LD), circular monomer (CM), linear trimer (LT), circular dimer (CD) and circular trimer (CT). The leftmost lane (labeled L) shows ligation with excess ligase, where almost all DNA is converted to circular products showing that it is at least 95% ligatable. The remaining lanes show a ligation time course, and are labeled with the reaction time in minutes. (b) A plot of the ratio 2M0C(t)/D(t) created from the gel shown in (a). The intercept of this plot at t = 0 gives the J factor measured in this experiment. The LM band shows some sample impurity, which we measured to be <5%.
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Figure 3: Example gel showing a ligation time course for the 116o sequence at a concentration of 0.33 nM ligated at 37°C with 50 U/ml T4 ligase. (a) Gel image showing both linearly and circularly ligated products. The bands in the gel are (from bottom to top) linear monomer (LM), linear dimer (LD), circular monomer (CM), linear trimer (LT), circular dimer (CD) and circular trimer (CT). The leftmost lane (labeled L) shows ligation with excess ligase, where almost all DNA is converted to circular products showing that it is at least 95% ligatable. The remaining lanes show a ligation time course, and are labeled with the reaction time in minutes. (b) A plot of the ratio 2M0C(t)/D(t) created from the gel shown in (a). The intercept of this plot at t = 0 gives the J factor measured in this experiment. The LM band shows some sample impurity, which we measured to be <5%.

Mentions: The J factor is calculated from cyclization experiments using (6):3where M0 is the initial concentration of the linear monomer of our DNA molecule, C(t) is the concentration of cyclized monomer, which is one molecule ligated to itself forming a closed loop and D(t) is the combined concentration of linear dimers and dimer circles, which may form directly from linear dimers. Since the only difference between the reactions that generate circular monomer and linear dimer is that for the former case, the DNA must bend into a closed loop while in the latter case it does not, the J factor gives a direct measure of DNA flexibility. Figure 3 shows an example of how the J factor is calculated from our experiments.Figure 3.


The flexibility of locally melted DNA.

Forties RA, Bundschuh R, Poirier MG - Nucleic Acids Res. (2009)

Example gel showing a ligation time course for the 116o sequence at a concentration of 0.33 nM ligated at 37°C with 50 U/ml T4 ligase. (a) Gel image showing both linearly and circularly ligated products. The bands in the gel are (from bottom to top) linear monomer (LM), linear dimer (LD), circular monomer (CM), linear trimer (LT), circular dimer (CD) and circular trimer (CT). The leftmost lane (labeled L) shows ligation with excess ligase, where almost all DNA is converted to circular products showing that it is at least 95% ligatable. The remaining lanes show a ligation time course, and are labeled with the reaction time in minutes. (b) A plot of the ratio 2M0C(t)/D(t) created from the gel shown in (a). The intercept of this plot at t = 0 gives the J factor measured in this experiment. The LM band shows some sample impurity, which we measured to be <5%.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724272&req=5

Figure 3: Example gel showing a ligation time course for the 116o sequence at a concentration of 0.33 nM ligated at 37°C with 50 U/ml T4 ligase. (a) Gel image showing both linearly and circularly ligated products. The bands in the gel are (from bottom to top) linear monomer (LM), linear dimer (LD), circular monomer (CM), linear trimer (LT), circular dimer (CD) and circular trimer (CT). The leftmost lane (labeled L) shows ligation with excess ligase, where almost all DNA is converted to circular products showing that it is at least 95% ligatable. The remaining lanes show a ligation time course, and are labeled with the reaction time in minutes. (b) A plot of the ratio 2M0C(t)/D(t) created from the gel shown in (a). The intercept of this plot at t = 0 gives the J factor measured in this experiment. The LM band shows some sample impurity, which we measured to be <5%.
Mentions: The J factor is calculated from cyclization experiments using (6):3where M0 is the initial concentration of the linear monomer of our DNA molecule, C(t) is the concentration of cyclized monomer, which is one molecule ligated to itself forming a closed loop and D(t) is the combined concentration of linear dimers and dimer circles, which may form directly from linear dimers. Since the only difference between the reactions that generate circular monomer and linear dimer is that for the former case, the DNA must bend into a closed loop while in the latter case it does not, the J factor gives a direct measure of DNA flexibility. Figure 3 shows an example of how the J factor is calculated from our experiments.Figure 3.

Bottom Line: One proposed explanation suggests that local melting of a few base pairs introduces flexible hinges.We have expanded this model to incorporate sequence and temperature dependence of the local melting, and tested it for three sequences at temperatures from 23 degrees C to 42 degrees C.We find that small melted bubbles are significantly more flexible than double-stranded DNA and can alter DNA flexibility at physiological temperatures.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, The Ohio State University, 191 West Woodruff Avenue, Columbus, OH 43210-1117, USA.

ABSTRACT
Protein-bound duplex DNA is often bent or kinked. Yet, quantification of intrinsic DNA bending that might lead to such protein interactions remains enigmatic. DNA cyclization experiments have indicated that DNA may form sharp bends more easily than predicted by the established worm-like chain (WLC) model. One proposed explanation suggests that local melting of a few base pairs introduces flexible hinges. We have expanded this model to incorporate sequence and temperature dependence of the local melting, and tested it for three sequences at temperatures from 23 degrees C to 42 degrees C. We find that small melted bubbles are significantly more flexible than double-stranded DNA and can alter DNA flexibility at physiological temperatures. However, these bubbles are not flexible enough to explain the recently observed very sharp bends in DNA.

Show MeSH
Related in: MedlinePlus