Light scattering studies of supercoiled and nicked DNA

Abstract

Static and dynamic light scattering measurements were made of solutions of pGem1a plasmids (3730 base pairs) in the relaxed circular (nicked) and supercoiled forms. The static structure factor and the spectrum of decay modes in the autocorrelation function were examined in order to determine the salient differences between the behaviors of nicked DNA and supercoiled DNA. The concentrations studied are within the dilute regime, which is to say that the structure and dynamics of an isolated DNA molecule were probed. Static light scattering measurements yielded estimates for the molecular weight M, second virial coefficient A2, and radius of gyration RG. For the nicked DNA, M = (2.8 +/- 0.4) x 10(6) g/mol, A2 = (0.9 +/- 0.2) x 10(-3) mol cm3/g2, and RG = 90 +/- 3 nm were obtained. For the supercoiled DNA, M = (2.5 +/- 0.4) x 10(6) g/mol, A2 = (1.2 +/- 0.2) x 10(-3) mol cm3/g2, and RG = 82 +/- 2.5 nm were obtained. The static structure factors for the nicked and supercoiled DNA were found to superpose when they were scaled by the radius of gyration. The intrinsic stiffness of DNA was evident in the static light scattering data. Homodyne intensity autocorrelation functions were collected for both DNAs at several angles, or scattering vectors. At the smallest scattering vectors the probe size was comparable to the longest intramolecular distance, while at the largest scattering vectors the probe size was smaller than the persistence length of the DNA. Values of the self-diffusion coefficients D were obtained from the low-angle data. For the DNA, D = (2.9 +/- 0.3) x 10(-8) cm2/s, and for the supercoiled DNA, D = (4.11 +/- 0.21) x 10(-8) cm2/s. The contribution to the correlation function from the internal dynamics of the DNA was seen to result in a strictly bimodal decay function. The rates of the faster mode gamma int, reached plateau values at low angles. For the nicked DNA, gamma int = 2500 +/- 500 s-1, and for the supercoiled DNA, gamma int = 5000 +/- 500 s-1. These rates correspond to the slowest internal relaxation modes of the DNAs. The dependence of the relaxation rates on scattering vector was monitored with the aid of cumulants analysis and compared with theoretical predictions for the semiflexible ring molecule. The internal mode rates and the dependence of the cumulants moments reflected the difference between the nicked DNA and the supercoiled DNA dynamical behavior. The supercoiled DNA behavior seen here indicates that conformational dynamics might play a larger role in DNA behavior than is suggested by the notion of a branched interwound structure.