PREPARATION AND CHARACTERIZATION OF FORM-V DNA, THE DUPLEX DNA RESULTING FROM ASSOCIATION OF COMPLEMENTARY, CIRCULAR SINGLE-STRANDED-DNA
PREPARATION AND CHARACTERIZATION OF FORM-V DNA, THE DUPLEX DNA RESULTING FROM ASSOCIATION OF COMPLEMENTARY, CIRCULAR SINGLE-STRANDED-DNA
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DOI：
10.1016/0022-2836(79)90299-7
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发表时间：
1979-01-01
影响因子：
5.6
通讯作者：
WEISSMANN, C
中科院分区：
文献类型：
--
作者：
STETTLER, UH;WEBER, H;WEISSMANN, C
在杂交条件下，[细菌]质粒P.beta.G或噬菌体PM2 DNA的互补环状单链，而不是单链vphi.X174 DNA的互补环状单链相互作用，产生双链复合体。这种DNA被称为V型，具有明确的物理化学性质。它在中性蔗糖梯度中以锐峰的形式沉淀；它在琼脂糖凝胶中的电泳率介于共价封闭的(形式I)和变性的DNA之间。通过EM分析，形式V表现为与形式I无法区分的高度折叠的双链分子。增加溴化乙锭的浓度导致形式I DNA的松弛和回缩对形式V没有类似的影响。在260 nm处，P.beta.G DNA的减色率为18.6%，而P.beta.G形式II DNA的减色率为23.4%，单链vphi.X174 DNA的减色率为10.5%。V型的热熔化是非合作的，吸光度逐渐增加，与单链DNA相似。V型DNA的圆二色谱不同于I型、环形刻痕(II型)和单链vphi.X174 DNA的圆二色谱，它在295 nm处有一个负带，主正带从273 nm移动到266 nm。形式V显然由右手沃森-克里克型双螺旋组成，这种双螺旋由相同数量的左手双螺旋和负超级线圈补偿。我们不能确定左手双链转动是通过碱基堆积和氢键来稳定的，例如在其他人描述的模型中，还是它们仅仅是补偿性转动而没有固有的稳定性。
Complementary circular single strands of [bacterial] plasmid P.beta.G or bacteriophage PM2 DNA but not of single-stranded .vphi.X174 DNA associate under hybridisation conditions, giving rise to a 2-stranded complex. This DNA, which is called form V, has well-defined physico-chemical properties. It sediments as a sharp peak in neutral sucrose gradients; its electrophoretic mobility in agarose gels is between that of covalently closed (form I) and denatured DNA. By EM, form V appears as highly folded duplex molecules indistinguishable from form I. Increasing concentrations of ethidium bromide which lead to relaxation and recoiling of form I DNA have no comparable effect upon form V. At 260 nm form V P.beta.G DNA has a hypochromicity of 18.6%, as compared to 23.4% in the case of P.beta.G form II DNA and 10.5% in the case of single-stranded .vphi.X174 DNA. The thermal melting of form V is non-cooperative with gradual increase in absorbance similar to that of single-stranded DNA. The circular dichroism spectrum of form V DNA differs from that of form I, circular nicked (form II) and single-stranded .vphi.X174 DNA in that it shows a negative band at 295 nm and a shift for the main positive band from 273 to 266 nm. Form V apparently consists of right-handed Watson-Crick type double-helices which are compensated by an equal number of left-handed duplex turns and negative supercoils. We cannot decide whether left-handed duplex turns are stabilized by base-stacking and H bonding, as for example in the models described by others, or whether they are merely compensatory turns without inherent stability.