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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

Rate of ligation versus concentration of T4 ligase at 37°C. The open circles show data from the 116o sequence, and the closed diamonds show data from the 116cl sequence. (a) The rate of formation of circular monomer increases linearly for all concentrations of ligase. (b) At high ligase concentration, the rate of dimer formation no longer increases linearly with ligase concentration. We find that ligation is linear up to 400 U/ml of ligase at 37°C, and therefore use ≤100 U/ml for our experiments at this temperature.
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Figure 2: Rate of ligation versus concentration of T4 ligase at 37°C. The open circles show data from the 116o sequence, and the closed diamonds show data from the 116cl sequence. (a) The rate of formation of circular monomer increases linearly for all concentrations of ligase. (b) At high ligase concentration, the rate of dimer formation no longer increases linearly with ligase concentration. We find that ligation is linear up to 400 U/ml of ligase at 37°C, and therefore use ≤100 U/ml for our experiments at this temperature.

Mentions: Cyclization experiments are performed using T4 DNA ligase (400 U/μl; NEB) in its standard buffer, with 0.1 mg/ml bovine serum albumin (BSA) (10 mg/ml; NEB) added. Cy5 labeled, HindIII-digested DNA is added at a concentration of 0.33 nM for 116-bp sequences and 10 nM for the 200-bp sequence. T4 ligase is added in concentrations of 25–100 U/ml, over a range of temperatures from 23°C to 42°C. Ligation is found to be linear for ligase concentrations <100 U/ml at 23°C, in agreement with previous studies (8). Figure 2 shows that we also find that ligation is linear for ligase concentrations up to 400 U/ml at 37°C. Ligase activity is quenched by increasing the ethylenediaminetetraacetic acid (EDTA) concentration to 0.05 M and samples are digested with proteinase K (Invitrogen) for 20 min at 65°C to inactivate ligase. We repeat experiments at several T4 ligase concentrations to ensure reproducibility. For the 116-bp sequences samples are concentrated by precipitation with linear polyacrylamide (32). Samples are visualized on 6% polyacrylamide gels. The gels are then imaged using a Typhoon Trio imager (GE Healthcare) set to detect the Cy5-labeled DNA in order to determine the concentration of the different ligation products.Figure 2.


The flexibility of locally melted DNA.

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

Rate of ligation versus concentration of T4 ligase at 37°C. The open circles show data from the 116o sequence, and the closed diamonds show data from the 116cl sequence. (a) The rate of formation of circular monomer increases linearly for all concentrations of ligase. (b) At high ligase concentration, the rate of dimer formation no longer increases linearly with ligase concentration. We find that ligation is linear up to 400 U/ml of ligase at 37°C, and therefore use ≤100 U/ml for our experiments at this temperature.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Rate of ligation versus concentration of T4 ligase at 37°C. The open circles show data from the 116o sequence, and the closed diamonds show data from the 116cl sequence. (a) The rate of formation of circular monomer increases linearly for all concentrations of ligase. (b) At high ligase concentration, the rate of dimer formation no longer increases linearly with ligase concentration. We find that ligation is linear up to 400 U/ml of ligase at 37°C, and therefore use ≤100 U/ml for our experiments at this temperature.
Mentions: Cyclization experiments are performed using T4 DNA ligase (400 U/μl; NEB) in its standard buffer, with 0.1 mg/ml bovine serum albumin (BSA) (10 mg/ml; NEB) added. Cy5 labeled, HindIII-digested DNA is added at a concentration of 0.33 nM for 116-bp sequences and 10 nM for the 200-bp sequence. T4 ligase is added in concentrations of 25–100 U/ml, over a range of temperatures from 23°C to 42°C. Ligation is found to be linear for ligase concentrations <100 U/ml at 23°C, in agreement with previous studies (8). Figure 2 shows that we also find that ligation is linear for ligase concentrations up to 400 U/ml at 37°C. Ligase activity is quenched by increasing the ethylenediaminetetraacetic acid (EDTA) concentration to 0.05 M and samples are digested with proteinase K (Invitrogen) for 20 min at 65°C to inactivate ligase. We repeat experiments at several T4 ligase concentrations to ensure reproducibility. For the 116-bp sequences samples are concentrated by precipitation with linear polyacrylamide (32). Samples are visualized on 6% polyacrylamide gels. The gels are then imaged using a Typhoon Trio imager (GE Healthcare) set to detect the Cy5-labeled DNA in order to determine the concentration of the different ligation products.Figure 2.

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