Effect of pH on the overstretching transition of double-stranded DNA: Evidence of force-induced DNA melting
Williams, Mark C
ABSTRACT When a single molecule of double-stranded DNA is stretched beyond its B-form contour length, the measured force shows a highly cooperative overstretching transition. We have investigated the source of this transition by altering helix stability with solution pH. As solution pH was increased from pH 6.0 to pH 10.6 in 250 mM NaCI, the overstretching transition force decreased from 67.0 +/- 0.8 pN to 56.2 +/- 0.8 pN, whereas the transition width remained nearly constant. As the pH was lowered from pH 6.0 to pH 3.1, the overstretching force decreased from 67.0 +/- 0.8 pN to 47.0 +/- 1.0 pN, but the transition width increased from 3.0 +/- 0.6 pN to 16.0 +/- 3 pN. These results quantitatively agree with a model that asserts that DNA strand dissociation, or melting, occurs during the overstretching transition.
INTRODUCTION
By stretching single molecules of DNA using optical tweezers or atomic force microscopy (AFM), a number of investigators have shown that DNA exhibits an unusual elastic behavior (Cluzel et al., 1996; Rief et al., 1999; Smith et al., 1996). These experiments move one end of a DNA molecule while measuring the force on the opposite end through means of an optical trap or AFM tip. The resulting force-extension curve is then used to describe molecular behavior under various solution conditions. At ~60-70 pN, the force-extension curve for double-stranded DNA (dsDNA) exhibits a plateau, indicating that the DNA can be elongated with very little additional force. This cooperative overstretch transition continues until the molecule is stretched to 1.7 times its B-form contour length, where the force increases rapidly and matches the force-extension curve of single-stranded DNA (ssDNA).
The overstretching transition has been attributed to a secondary structure transition from B-form to S-form DNA (Cluzel et al., 1996). The structure of S-form DNA is not well understood, but molecular models have been proposed (Cluzel et al., 1996; Konrad and Bolonick, 1996; Kosikov et al., 1999; Lebrun and Lavery, 1996) in which DNA unwinds to form a ladder-like structure or in which the DNA molecule forms a reduced-radius fiber. These models assume that the bases remain paired during the overstretching transition and that the transition is reversible. Although they are able to reproduce the observed plateau in the force-extension curve, the models are not able to predict the transition force or its width. The plateau is attributed to a transition in which only the base-stacking interactions are lost. A good fit to the observed data can be obtained using a two-state transition model (Ahsan et al., 1998; Cizeau and Viovy, 1997) or a simple elastic model (Haijun et al., 1999). However, because they are unable to predict the overstretching transition force, it is not clear how the models are related to the actual structural changes that occur as the DNA is stretched.
Rouzina and Bloomfield (2000a,b) have developed a theory that predicts that strand dissociation, or melting, occurs during the overstretch transition. The theory quantitatively accounts for the observed overstretching force and the slope of the overstretching force as a function of extension. It also predicts that the overstretching force will decrease if conditions are changed to destabilize the helix.
To test this theory, we have used an optical tweezers instrument to measure the overstretching force and the width of the overstretching transition as a function of pH. Because extremes of high or low pH destabilize the helix and lower the thermal melting point of DNA (Lando et al., 1994), the theory predicts that the overstretching force will decrease in both low and high pH. Our data show that the overstretching force and thermal melting point as a function of pH exhibit similar trends and that the model of forceinduced melting accurately describes the dependence of the overstretching force on pH.
We thank Prof. Matthew Tirrell and the University of Minnesota Center for Interfacial Engineering for funding and assistance in starting the optical tweezers project. We are grateful to Drs. Steve Smith and Christoph Baumann for help with protocols and instrument-building advice. We also thank Dori Henderson for taking the time to make a number of glass micropipettes for use in our experiments.
Funding for this project was provided by grants from the National Institutes of Health (GM28093) and National Science Foundation (MCB9728165).
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Mark C. Williams, Jay R. Wenner, Ioulia Rouzina, and Victor A. Bloomfield
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Saint Paul, Minnesota 55108 USA
Received for publication 16 June 2000 and in final form 22 November 2000.
Address reprint requests to Dr. Victor A. Bloomfield, Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Saint Paul, MN 55108. Tel.: 612-625-2268; Fax: 612-625-5780; E-mail: victor.a.bloomfield-1@tc.umn.edu.
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