Lu Y, Weers B, Stellwagen N C
Department of Biochemistry, University of Iowa, Iowa City, IA 52242, USA.
Biopolymers. 2001;61(4):261-75. doi: 10.1002/bip.10151.
DNA restriction fragments ranging from 79 to 789 base pairs in length have been characterized by transient electric birefringence (TEB) measurements at various temperatures between 4 and 43 degrees C. The DNA fragments do not contain runs of four or more adenine residues in a row and migrate with normal electrophoretic mobilities in polyacrylamide gels, indicating that they are not intrinsically curved or bent. The low ionic strength buffers used for the measurements contained 1 mM Tris Cl, pH 8.0, EDTA, and variable concentrations of Na(+) or Mg(2+) ions. The rotational relaxation times were obtained by fitting the TEB field-free decay signals with a nonlinear least-squared fitting program; the decay of the birefringence was monoexponential for fragments < or = 241 base pair (bp) in length and multiexponential for larger fragments. The terminal relaxation times, characteristic of the end-over-end rotation of the DNA molecules, were then used to determine the persistence length (p) and hydrodynamic radius (r) of DNA as a function of temperature and ionic strength, using several different hydrodynamic models. The specific values obtained for p and r are model dependent. The wormlike chain model of P. J. Hagerman and B. H. Zimm (Biopolymers 1981, Vol. 20, pp. 1481-1502) combined with the revised Broersma equation (J. Newman et al., Journal of Mol Biol 1997, Vol. 116, pp. 593-606) appears to be the most suitable for describing the flexibility of DNA in low ionic strength solutions. The values of p and r obtained from the global least squares fitting of this equation are independent of DNA length, and the deviations of the individual values from the average are reasonably small. The consensus r value calculated for DNA in various low ionic strength solutions containing 1 mM Tris buffer is 14.7 +/- 0.4 A at 20 degrees C. The consensus p values decrease from 814 approximately 564 A in solutions containing 1 mM Tris buffer plus 0.2-1 mM NaCl and decrease still further to 440 A in solutions containing 0.2 mM Mg(2+) ions. The persistence length exhibits a shallow maximum at 20 degrees C and decreases slowly upon either increasing or decreasing the temperature, regardless of the model used to fit the data. By contrast, the consensus values of the hydrodynamic radius are independent of temperature. The calculated persistence lengths and hydrodynamic radii are compared with other data in the literature.
通过在4至43摄氏度之间的不同温度下进行瞬态电双折射(TEB)测量,对长度在79至789个碱基对之间的DNA限制性片段进行了表征。这些DNA片段不包含连续四个或更多个腺嘌呤残基,并且在聚丙烯酰胺凝胶中以正常的电泳迁移率迁移,这表明它们并非固有弯曲或弯折的。用于测量的低离子强度缓冲液含有1 mM Tris Cl,pH 8.0,EDTA以及可变浓度的Na(+)或Mg(2+)离子。通过使用非线性最小二乘拟合程序对TEB无场衰减信号进行拟合来获得旋转弛豫时间;对于长度小于或等于241个碱基对(bp)的片段,双折射衰减是单指数的,而对于较大的片段则是多指数的。然后,使用几种不同的流体动力学模型,将DNA分子端对端旋转所特有的末端弛豫时间用于确定DNA的持久长度(p)和流体动力学半径(r),它们是温度和离子强度的函数。所获得的p和r的具体值取决于模型。PJ Hagerman和BH Zimm的蠕虫状链模型(《生物聚合物》1981年,第20卷,第1481 - 1502页)与修订后的布罗斯马方程(J. Newman等人,《分子生物学杂志》1997年,第116卷,第593 - 606页)相结合,似乎最适合描述低离子强度溶液中DNA的柔韧性。从该方程的全局最小二乘拟合中获得的p和r值与DNA长度无关,并且各个值与平均值的偏差相当小。在含有1 mM Tris缓冲液的各种低离子强度溶液中计算得到的DNA的共识r值在20摄氏度时为14.7 +/- 0.4 Å。在含有1 mM Tris缓冲液加0.2 - 1 mM NaCl的溶液中,共识p值从约814 Å降至564 Å,而在含有0.2 mM Mg(2+)离子的溶液中则进一步降至440 Å。无论用于拟合数据的模型如何,持久长度在20摄氏度时呈现出一个浅的最大值,并且在温度升高或降低时都会缓慢下降。相比之下,流体动力学半径的共识值与温度无关。将计算得到的持久长度和流体动力学半径与文献中的其他数据进行了比较。