Wu P G, Fujimoto B S, Song L, Schurr J M
Department of Chemistry, BG-10 University of Washington, Seattle 98195.
Biophys Chem. 1991 Dec;41(3):217-36. doi: 10.1016/0301-4622(91)85038-r.
The torsion elastic constants (alpha) of linear pBR322 (4363 bp) and pUC8 (2717 bp) DNAs and supercoiled pBR322 and pJMSII (4375 bp) DNAs are measured in 0.1 M NaCl as a function of added ethidium/base-pair (EB/BP) ratio by studying the fluorescence polarization anisotropy (FPA) of the intercalated ethidium. The time-resolved FPA is measured by using a picosecond dye laser for excitation and time-correlated single photon counting detection. Previously developed theory for the emission anisotropy is generalized to incorporate rotations of the transition dipole due to excitation transfer. The excitation transfers are simulated by a Monte Carlo procedure (Genest et al., Biophys. Chem. 1 (1974) 266-278) and the consequent rotations of the transition dipole are superposed on the Brownian rotations. After accounting for excitation transfer, the torsion constants of the linear DNAs are found to be essentially independent of intercalated ethidium up to a binding ratio r = 0.10 dye/bp. Dynamic light scattering measurements on linear pUC8 DNA confirm that the torsion constant is independent of binding ratio up to r = 0.20 dye/bp. If alpha d denotes the torsion constant between ethidium and a base-pair, and alpha 0 that between two base-pairs, then our data imply that alpha d/alpha 0 lies in the range 0.65 to 1.64 with a most probable value of 1.0. The torsion constants of supercoiled DNAs decrease substantially with increasing binding ratio even after accounting for excitation transfer. At the binding ratio r* = 0.064, where the superhelix density vanishes and superhelical strain is completely relaxed, the torsion constant of the supercoiled pBR322 DNA/dye complex lies below that of the corresponding linear DNA/dye complex by about 30%. This contradicts the conventional view according to which linear, nicked circular, and supercoiled DNA/dye complexes with r = r* should coexist with the same concentration of free dye, display the same distribution of bound dye, and exhibit identical secondary structures, twisting and bending rigidities, and FPA dynamics. These and other observations imply the existence of metastable secondary structure in freshly relaxed supercoiled DNAs. A tentative explanation is presented for these and other unexpected observations on supercoiled DNAs.
通过研究嵌入溴化乙锭的荧光偏振各向异性（FPA），在0.1 M NaCl中测量线性pBR322（4363 bp）和pUC8（2717 bp）DNA以及超螺旋pBR322和pJMSII（4375 bp）DNA的扭转弹性常数（α）与添加的溴化乙锭/碱基对（EB/BP）比率的函数关系。使用皮秒染料激光器进行激发并通过时间相关单光子计数检测来测量时间分辨FPA。先前开发的发射各向异性理论被推广以纳入由于激发转移导致的跃迁偶极子的旋转。通过蒙特卡罗程序（Genest等人，《生物物理化学》1（1974）266 - 278）模拟激发转移，并将由此产生的跃迁偶极子的旋转叠加在布朗旋转上。在考虑激发转移后，发现线性DNA的扭转常数在结合比率r = 0.10染料/碱基对之前基本上与嵌入的溴化乙锭无关。对线性pUC8 DNA的动态光散射测量证实，扭转常数在结合比率r = 0.20染料/碱基对之前与结合比率无关。如果αd表示溴化乙锭与一个碱基对之间的扭转常数，α0表示两个碱基对之间的扭转常数，那么我们的数据表明αd/α0在0.65至1.64范围内，最可能值为1.0。即使考虑了激发转移，超螺旋DNA的扭转常数也随着结合比率的增加而大幅降低。在结合比率r* = 0.064时，超螺旋密度消失且超螺旋应变完全松弛，超螺旋pBR322 DNA/染料复合物的扭转常数比相应的线性DNA/染料复合物低约30%。这与传统观点相矛盾，根据传统观点，r = r*的线性、带切口的环状和超螺旋DNA/染料复合物应与相同浓度的游离染料共存，显示相同的结合染料分布，并表现出相同的二级结构、扭曲和弯曲刚性以及FPA动力学。这些以及其他观察结果表明，新松弛的超螺旋DNA中存在亚稳二级结构。针对这些以及关于超螺旋DNA的其他意外观察结果提出了一个初步解释。
