Structure and dynamics of curved DNA fragments in solution: Evidence for slow modes of configurational transitions

Abstract

DNA fragments with unusually low electrophoretic mobility due to intrinsic curvature have been analyzed by comparison of electrooptical data with results of hydrodynamic simulations. Electrooptical data have been collected for three fragments with 161, 196 and 399 base pairs derived from the DNA of Chironomus thummi thummi as repetitive elements by Alu I restriction. The dichroism decay time constants reflecting overall rotational diffusion, the bending time constants and the bending amplitudes measured at low salt concentrations (2.4 m M Na + and 100 μ M Mg 2+ are rather close to those observed for standard DNA fragments. At high salt concentration (0.1 M Na 2+ and 10 m M Mg 2+) the temperature dependence of the overall rotational time constants indicates a slightly increased degree of curvature at low temperature (2°C). The experimental data are complemented by hydrodynamic simulations based on predictions of DNA trajectories given by Bolshoy et al. [ Proc. Natl. Acad. Sci. USA 88 (1991) 2312]. These trajectories are converted into bead models, which are then subjected to thermal fluctuations using a Monte Carlo procedure. For standard values of the persistence length and the torsional flexibility, thermal fluctuations induce considerable variations of the equilibrium curvature. As a first attempt to find conditions where the predicted trajectories are consistent with our hydrodynamic data, we tested a model with a high internal mobility, which has been commonly applied for standard DNA fragments. However, the overall rotational time constants predicted for this case are clearly smaller than the observed ones, even at high values of the persistence length. Then, we simulated time constants in the limit of low internal mobility by calculation of electrooptical transients for large numbers of individual configurations. The average of these transients could be fitted by two exponentials at high accuracy, although the simulations led to broad distributions of configurations. In this respect the simulated curves are very similar to the experimental ones. For standard values of the persistence length and of the torsional flexibility, the large time constants τ 2, reflecting overall rotational diffusion, are still smaller than the experimental ones. τ 2-values simulated as a function of the persistence length p show a maximum, which appears at p ≈ 1000 Å for the Alu-fragments. The τ 2-values simulated at these maxima are consistent with the experimental ones within the limits of accuracy. Thus, provided that the curvature has been estimated correctly by the model based on gel mobilities and on circularization experiments curved DNA fragments show a relatively low rate of the internal dynamics and also appear to be less flexible than standard DNA's with respect to the dynamic persistence. The difference in the dynamic persistence is negligible, however, if the apparent persistence length of standard DNA has a major contribution from intrinsic curvature, corresponding to an average static persistence length of about 800 Å. In summary, our results indicate that the “deviations from linearity” of our curved fragments are not much different from those of standard DNA's; however, our results are consistent with the view that curved DNA fragments are “curved” preferentially in one direction with relatively slow modes of configurational transitions and with a relatively high rigidity, whereas standard DNA is subject to bending by thermal motion in all directions with (almost) equal probability.