Ringquist S, Shinn R, Hanlon S
Department of Biological Chemistry, University of Illinois College of Medicine, Chicago 60612.
Biochemistry. 1989 Feb 7;28(3):1076-85. doi: 10.1021/bi00429a023.
Changes in linking number and the apparent winding angle of pBR322 DNA have been evaluated in mixed ethanol-water solvents containing either Na or Mg as the major counterion contributing to the electrostatic shielding of the duplex. The average number of superhelical turns (tau) produced in the standard electrophoresis buffer (Tris-borate-EDTA, pH 8.0) by the transfer of DNA, relaxed in 200 mM NaCl, 10 mM NaH2PO4/Na2HPO4, and 2 mM EDTA, pH 7, by calf thymus topoisomerase or ligated in 6.6 mM MgCl2, 1 mM KCl, 1 mM ATP, 1 mM dithiothreitol, and 66 mM Tris, pH 7.6, by T4 ligase, was determined as a function of the EtOH concentration. At low enzyme concentrations, the tau values became increasingly more positive in the presence of both cations as the ethanol concentration increased, indicating that the duplex structure was overwound in the ethanol solvents. Winding angle changes between 0 and 20% ethanol, calculated from these values of tau, exhibited the same correlations with CD spectral properties as had been previously observed for 100% aqueous systems containing monovalent cations [Kilkuskie, R., Wood, N., Shinn, R., Ringquist, S., & Hanlon, S. (1988) Biochemistry 27, 4377-4386]. The results at higher concentrations of ethanol (25-30%), however, were anomalous for the Mg-ligase system. The anomalies increased with higher ethanol, ligase, or Mg concentration. Gel run under these conditions showed enhanced concentrations of slow-moving components, indicative of ligation of intermolecular associated DNA species. At a 10-fold higher level of ligase, ethanol appeared to unwind the duplex, confirming the results of Lee, Mizusawa, and Kakefuda [(1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2838-2842]. All of these anomalies occur under solvent conditions which are close to conditions which produce a heterogeneous dispersion of sedimenting species in ultracentrifugal experiments and compact rodlike structures, visualized by electron microscopy. The circular dichroism spectra at the onset of the formation of these structures show the characteristics of a chirally packed array of DNA duplexes. The reversal of the trend of the ethanol effect on linking number at higher enzyme and Mg(II) concentrations can be most easily explained by the promotion of the condensation phenomenon by either the ligase or a contaminating factor in the preparation. We suggest that the anomalies in the linking number and winding angle values are due to either ligation of chirally bent DNA species or a change in the helical period as the linear DNA adapts to the conformation required for collapse.(ABSTRACT TRUNCATED AT 400 WORDS)
在以Na或Mg作为主要抗衡离子以对双链体进行静电屏蔽的乙醇 - 水混合溶剂中，评估了pBR322 DNA的连环数和表观缠绕角的变化。通过小牛胸腺拓扑异构酶将在200 mM NaCl、10 mM NaH2PO4 / Na2HPO4和2 mM EDTA（pH 7）中松弛的DNA转移至标准电泳缓冲液（Tris - 硼酸盐 - EDTA，pH 8.0）中，或者通过T4连接酶在6.6 mM MgCl2、1 mM KCl、1 mM ATP、1 mM二硫苏糖醇和66 mM Tris（pH 7.6）中连接产生的超螺旋匝数（τ）的平均值，被确定为乙醇浓度的函数。在低酶浓度下，随着乙醇浓度增加，在两种阳离子存在时τ值变得越来越正，表明双链体结构在乙醇溶剂中过度缠绕。根据这些τ值计算出的0至20%乙醇之间的缠绕角变化，与CD光谱特性呈现出与先前在含有单价阳离子的100%水性体系中观察到的相同相关性[Kilkuskie, R., Wood, N., Shinn, R., Ringquist, S., & Hanlon, S. (1988) Biochemistry 27, 4377 - 4386]。然而，在较高乙醇浓度（25 - 30%）下，Mg - 连接酶体系的结果异常。随着乙醇、连接酶或Mg浓度升高，异常情况增加。在这些条件下进行的凝胶电泳显示慢迁移组分的浓度增加，表明分子间相关DNA物种发生了连接。在连接酶浓度高10倍的情况下，乙醇似乎使双链体解缠，证实了Lee、Mizusawa和Kakefuda [(1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2838 - 2842]的结果。所有这些异常都发生在接近在超速离心实验中产生沉降物种的异质分散和电子显微镜下可见的紧密棒状结构的溶剂条件下。这些结构形成开始时的圆二色光谱显示出DNA双链体手性堆积阵列的特征。在较高酶和Mg(II)浓度下乙醇对连环数影响趋势的逆转，最容易通过连接酶或制剂中的污染因子促进凝聚现象来解释。我们认为连环数和缠绕角值的异常是由于手性弯曲DNA物种的连接或线性DNA适应塌陷所需构象时螺旋周期的变化所致。（摘要截短于400字）
