Fogg Jonathan M, Kolmakova Natalia, Rees Ian, Magonov Sergei, Hansma Helen, Perona John J, Zechiedrich E Lynn
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA.
J Phys Condens Matter. 2006 Apr 12;18(14):S145-S159. doi: 10.1088/0953-8984/18/14/S01.
Using λ-Int recombination in E. coli, we have generated milligram quantities of supercoiled minicircle DNA. Intramolecular Int recombination was efficient down to lengths ~254 bp. When nicked and religated in the presence of ethidium bromide, 339 bp minicircles adopted at least seven unique topoisomers that presumably correspond to ΔLk ranging from 0 to -6, which we purified individually. We used these minicircles, with unique ΔLk, to address the partition into twist and writhe as a function of ΔLk. Gel electrophoresis and atomic force microscopy revealed progressively higher writhe conformations in the presence of 10 mM CaCl(2) or MgCl(2). From simplistic calculations of the bending and twisting energies, we predict the elastic free energy of supercoiling for these minicircles to be lower than if the supercoiling was partitioned mainly into twist. The predicted writhe corresponds closely with that which we observed experimentally in the presence of divalent metal ions. However, in the absence of divalent metal ions only limited writhe was observed, demonstrating the importance of electrostatic effects on DNA structure, when the screening of charges on the DNA is weak. This study represents a unique insight into the supercoiling of minicircle DNA, with implications for DNA structure in general.
利用大肠杆菌中的λ-Int重组，我们已生成了毫克级的超螺旋微环DNA。分子内Int重组在长度约为254 bp时仍很高效。当在溴化乙锭存在下进行切口和重新连接时，339 bp的微环采用了至少七种独特的拓扑异构体，推测其对应于从0到-6的ΔLk，我们对其进行了单独纯化。我们使用这些具有独特ΔLk的微环来研究作为ΔLk函数的扭曲和缠绕分配情况。凝胶电泳和原子力显微镜显示，在存在10 mM CaCl₂或MgCl₂的情况下，缠绕构象逐渐增加。通过对弯曲和扭曲能量的简单计算，我们预测这些微环的超螺旋弹性自由能低于超螺旋主要分配到扭曲的情况。预测的缠绕与我们在二价金属离子存在下实验观察到的情况密切相关。然而，在没有二价金属离子的情况下，仅观察到有限的缠绕，这表明当DNA上电荷的屏蔽较弱时，静电效应对DNA结构的重要性。这项研究为微环DNA的超螺旋提供了独特的见解，对一般的DNA结构也有启示。
