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Volume 271, Number 46, Issue of November 15, 1996 pp. 29126-29135
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Microscopic DNA Flexibility Analysis
PROBING THE BASE COMPOSITION AND ION DEPENDENCE OF MINOR GROOVE COMPRESSION WITH AN ARTIFICIAL DNA BENDING AGENT

(Received for publication, May 30, 1996, and in revised form, September 4, 1996)

From the Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030

We have used an artificial DNA bending agent to monitor the local flexibility of the DNA helix as a function of Mg²⁺ cation concentration, sequence, and temperature. A DNA bending agent was constructed from a pair of triple helix-forming oligonucleotides connected by a flexible polymeric linker, which, when the linker is short enough, causes a bend in a minor groove region separating the two sites of triple helix formation. The unique aspect of this system is that, since the bent region is not in direct contact with the linker or the triple helix-forming oligonucleotides, the free energy reflecting the bendability of the minor helix groove can be estimated from a comparison of binding affinity between the bent and unbent triple helices. A binding competition experiment and association and dissociation kinetic assays executed at 37 °C in the presence of 10 mM Mg²⁺ have revealed an extremely small difference in binding affinity between bent (50°) and straight triple helices, suggesting that DNA flexibility with respect to minor groove compression is extremely high and virtually independent of the sequence of the distorted duplex. This unexpectedly small difference in binding affinity was detected over the temperature range from 25 to 65 °C, and over a Mg²⁺ concentration range from 0.3 to 10 mM. Thus, these findings provide evidence that DNA bendability for minor groove compression is inherently high and independent of DNA sequence, temperature, or a 30-fold variation of Mg²⁺ ion concentration.

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