Lau P P, Gray H B
Nucleic Acids Res. 1980 Feb 11;8(3):673-701. doi: 10.1093/nar/8.3.673.
Addition of the intercalating dye ethidium bromide (EtdBr) to a solution of alkali-denatured double-stranded closed circular PM2, PhiX174, or lambdab(2)b(5)c phage DNAs, under conditions such that the solution remains strongly alkaline, can result in the renaturation of up to 100% of the DNA upon neutralization of the solution. For a fixed time of incubation of the alkaline dye-containing solution before neutralization, there exists a minimum concentration of the dye below which no EtdBr-mediated renaturation is observed for each species of closed circular DNA examined. These minimum concentrations increase, for a given DNA, with increasing ionic strength and temperature. The kinetics of accumulation of forms renaturing upon neutralization of alkaline solutions, at fixed concentrations of dye and DNA, are dependent upon the molecular weight and superhelix density of the starting DNA. After extended periods of incubation at a fixed ionic strength and temperature, however, the profiles of percentage of DNA renatured as a function of ethidium concentration become very similar for all the closed circular DNAs tested and display a transition from an absence of dye-mediated renaturation to virtually 100% renaturation upon neutralization over a small range of dye concentration. Circular DNA containing one or more strand scissions remains strand-separated under all the conditions used to effect the renaturation of closed circular DNA. These findings indicate that configurations of closed circular DNA, in which at least some of the complementary bases are apposed, can be selectively stabilized and accumulate in the presence of ethidium in solutions containing 0.19 N hydroxide ion. The closed circular DNA of bacteriophage PM2 has properties distinct from those of the other DNAs of this study in that it has been shown to contain fractions which exist in the base-paired duplex form after neutralization of strongly alkaline solutions of this DNA incubated at ambient temperature, while no duplex DNA is observed after exposure to alkali and neutralization of solutions of closed circular DNA from other sources. (1,2) The fraction of denaturation-resistant PM2 DNA is shown in the present work to depend upon the temperature and time of incubation in alkali, but not upon the superhelix density of the starting DNA. PM2 closed circular DNA also behaves anomalously with respect to its kinetics of accumulation of forms renaturing upon neutralization of alkaline, EtdBr-containing solutions. Evidence is presented that the translocation of one of the strands of a closed circular molecule relative to the other, which is required for the molecule to exist in the denatured form at neutral pH, is a process to which PM2 DNA is less labile than the other closed circular DNAs of this study.
在溶液保持强碱性的条件下，向碱变性的双链闭环PM2、PhiX174或λb(2)b(5)c噬菌体DNA溶液中添加嵌入染料溴化乙锭（EtdBr），溶液中和后DNA的复性率可达100%。对于中和前含碱性染料溶液的固定孵育时间，每种所检测的闭环DNA都存在一个染料最低浓度，低于该浓度则未观察到EtdBr介导的复性。对于给定的DNA，这些最低浓度会随着离子强度和温度的升高而增加。在染料和DNA浓度固定的情况下，碱性溶液中和后复性形式积累的动力学取决于起始DNA的分子量和超螺旋密度。然而，在固定离子强度和温度下长时间孵育后，对于所有测试的闭环DNA，复性DNA百分比随溴化乙锭浓度变化的曲线变得非常相似，并且在染料浓度的小范围内显示出从无染料介导的复性到中和后几乎100%复性的转变。含有一个或多个链断裂的环状DNA在用于实现闭环DNA复性的所有条件下都保持链分离状态。这些发现表明，在含有0.19 N氢氧根离子的溶液中，在溴化乙锭存在的情况下，至少一些互补碱基并列的闭环DNA构型可以被选择性稳定并积累。噬菌体PM2的闭环DNA具有与本研究中其他DNA不同的特性，因为已表明它含有在室温下孵育该DNA的强碱性溶液中和后以碱基配对双链形式存在的组分，而在暴露于碱并中和来自其他来源的闭环DNA溶液后未观察到双链DNA。（1,2）在本研究中表明，抗变性的PMDNA部分取决于在碱中的孵育温度和时间，而不取决于起始DNA的超螺旋密度。PM2闭环DNA在含EtdBr的碱性溶液中和后复性形式积累的动力学方面也表现异常。有证据表明，闭环分子的一条链相对于另一条链的易位是该分子在中性pH下以变性形式存在所必需的过程，与本研究中的其他闭环DNA相比，PM2 DNA对该过程的稳定性较低。
