Hammermann M, Brun N, Klenin K V, May R, Tóth K, Langowski J
Biophysics of Macromolecules Division, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.
Biophys J. 1998 Dec;75(6):3057-63. doi: 10.1016/S0006-3495(98)77746-X.
Using small angle neutron scattering we have measured the static form factor of two different superhelical DNAs, p1868 (1868 bp) and pUC18 (2686 bp), in dilute aqueous solution at salt concentrations between 0 and 1.5 M Na+ in 10 mM Tris at 0% and 100% D2O. For both DNA molecules, the theoretical static form factor was also calculated from an ensemble of Monte Carlo configurations generated by a previously described model. Simulated and measured form factors of both DNAs showed the same behavior between 10 and 100 mM salt concentration: An undulation in the scattering curve at a momentum transfer q = 0.5 nm-1 present at lower concentration disappears above 100 mM. The position of the undulation corresponds to a distance of approximately 10-20 nm. This indicated a change in the DNA superhelix diameter, as the undulation is not present in the scattering curve of the relaxed DNA. From the measured scattering curves of superhelical DNA we estimated the superhelix diameter as a function of Na+ concentration by a quantitative comparison with the scattering curve of relaxed DNA. The ratio of the scattering curves of superhelical and relaxed DNA is very similar to the form factor of a pair of point scatterers. We concluded that the distance of this pair corresponds to the interstrand separation in the superhelix. The computed superhelix diameter of 16.0 +/- 0.9 nm at 10 mM decreased to 9.0 +/- 0.7 nm at 100 mM salt concentration. Measured and simulated scattering curves agreed almost quantitatively, therefore we also calculated the superhelix diameter from the simulated conformations. It decreased from 18.0 +/- 1.5 nm at 10 mM to 9.4 +/- 1.5 nm at 100 mM salt concentration. This value did not significantly change to lower values at higher Na+ concentration, in agreement with results obtained by electron microscopy, scanning force microscopy imaging in aqueous solution, and recent MC simulations, but in contrast to the observation of a lateral collapse of the DNA superhelix as indicated by cryo-electron microscopy studies.
我们使用小角中子散射技术，在0%和100% D₂O的10 mM Tris缓冲液中，于盐浓度介于0至1.5 M Na⁺之间的稀水溶液中，测量了两种不同超螺旋DNA（p1868，1868 bp；pUC18，2686 bp）的静态形状因子。对于这两种DNA分子，理论静态形状因子也根据先前描述的模型生成的蒙特卡洛构型系综进行了计算。两种DNA的模拟和测量形状因子在10至100 mM盐浓度之间表现出相同的行为：较低浓度下在动量转移q = 0.5 nm⁻¹处散射曲线中的波动在100 mM以上消失。波动的位置对应于约10 - 20 nm的距离。这表明DNA超螺旋直径发生了变化，因为在松弛DNA的散射曲线中不存在这种波动。通过与松弛DNA的散射曲线进行定量比较，我们从超螺旋DNA的测量散射曲线中估计了超螺旋直径作为Na⁺浓度的函数。超螺旋DNA和松弛DNA散射曲线的比值与一对点散射体的形状因子非常相似。我们得出结论，这一对点散射体的距离对应于超螺旋中的链间间距。在10 mM时计算得到的超螺旋直径为16.0 ± 0.9 nm，在100 mM盐浓度下降至9.0 ± 0.7 nm。测量和模拟的散射曲线几乎在数量上一致，因此我们也从模拟构象中计算了超螺旋直径。它从10 mM时的18.0 ± 1.5 nm降至100 mM盐浓度下的9.4 ± 1.5 nm。该值在较高Na⁺浓度下没有显著降低，这与电子显微镜、水溶液中的扫描力显微镜成像以及最近的蒙特卡洛模拟结果一致，但与低温电子显微镜研究表明的DNA超螺旋横向塌陷的观察结果相反。
