EFFECTS OF NA- COMPARISON OF SIMULATIONS WITH EXPERIMENTS( AND MG2+ ON THE STRUCTURES OF SUPERCOILED DNAS )

Recent cryo-electron microscopy (cryo-EM) results suggest that suffici ent NaCl concentration (greater than or similar to 0.1 M) and superhel ix density (greater than or similar to-0.05) cause circular DNAs to ad opt highly extended, tightly interwound configurations, in which the s trands are laterally contiguous along almost their entire length. Mill imolar levels of MgCl2 reportedly act synergistically with NaCl to pro duce similar conformations. However, Monte Carlo simulations with pure ly repulsive interduplex forces failed to reproduce such structures. I n the present work, solution measurements of particular physical prope rties were performed both to characterize the effects of Na+ and Mg2on DNA structure and to provide quantitative tests of Monte Carlo simu lations of circular DNAs. Supercoiled p30 delta DNAs in 10 mM Tris plu s 0, 0.122, and 0.1 M NaCl, and 0.1 M NaCl plus 4 mM Mg2+ were examine d by static and dynamic light scattering (LS and DLS), time-resolved f luorescence polarization anisotropy (FPA) of intercalated ethidium, an d circular dichroism (CD) spectroscopy. Upon addition of 0.122 M NaCl, the radius of gyration (R(g)) decreased substantially, which indicate s that p30 delta adopts a more compact structure. This contradicts the cryo-EM studies, where molecular extension and R(g) both increase upo n adding 0.1 M NaCl. In 0.1 M NaCl, the torsion constant measured by F PA is practically invariant to superhelix density, and the plateau dif fusion coefficient at large scattering vector (D-plat) is likewise nea rly the same at both relaxed and native superhelix densities. Such inv ariance is difficult to reconcile with any transition from relaxed cir cles to tightly interwound structures with laterally contiguous strand s. Metropolis Monte Carlo simulations were performed to generate canon ically distributed sets of structures, from which average D-0 values a nd scattered intensity ratios, [I(0)]/[I(k)], were calculated. Agreeme nt between simulations and experiments in regard to [I(0)]/[I(k)], D-0 and the supercoiling free energy, Delta G(sc)(Delta l), is remarkably good for the most extensively studied p30 delta samples. The simulate d structures exhibit no sign of very tight interwinding with extensive lateral contacts, but instead exhibit most probable superhelix diamet ers of 85 to 90 Angstrom. When 4 mM Mg2+ was added to native supercoil ed p30 delta in 0.1 M NaCl, R(g) decreased, D-0 increased, and the lon gest internal relaxation rate (1/tau(2)(0)) increased, all of which in dicate a further overall contraction of the molecular envelope. The to rsion constant exhibited a slight increase that is hardly statisticall y significant. In this case, agreement between the simulations and exp eriments was only semi-quantitative for most samples investigated, alt hough the predicted contraction was exhibited by all five samples of p 30 delta and one of pBR322 DNA. The simulated structures in 0.1 M NaCl plus 4 mM Mg2+ again showed no sign of extensive lateral contacts. A plausible explanation is proposed for the highly extended, tightly int erwound structures seen in cryo-EM, and explicitly tested by Monte Car lo simulations of a 1000 bp circular DNA at +25 and -50 degrees C. Str uctures identical to those seen in cryo-EM are in fact the equilibrium structures in the simulations at -50 degrees C, and the estimated tim e for equilibration (2.3 x 10(-6) second) is much smaller than the est imated time for vitrification (1 x 10(-4) second). (C) 1996 Academic P ress Limited