Dickie P, Morgan A R, McFadden G
Department of Biochemistry, University of Alberta, Edmonton, Canada.
J Mol Biol. 1988 May 5;201(1):19-30. doi: 10.1016/0022-2836(88)90435-4.
The variable positions of a branch-migrating cruciform junction in supercoiled plasmid DNA were mapped following cleavage of the DNA with bacteriophage T7 endonuclease I. T7 endonuclease I specifically cleaved, and thereby resolved, the Holliday junction existing at the base of the cruciform in the circular bacterial plasmid pSA1B.56A. Cruciform extrusion of cloned sequences in pSA1B.56A (containing a 322 base-pair inverted repeat insert composed of poxvirus telomeric sequences) topologically relaxed the plasmid substrate in vitro. Thus, numerous crossover positions were identified within the region of cloned sequences, reflecting the range of superhelical densities in the native plasmid preparation. Endonuclease I-sensitive crossover positions, mapped to both strands of the viral insert following the T7 endonuclease I digestion of either plasmid preparations or individual topoisomers, were regularly separated by approximately ten nucleotides. The appearance of sensitive crossovers every ten nucleotides corresponds to a change in linking difference (delta Lk) of +/- 2 in the circular core domain of the plasmid during branch point migration. In contrast, individual topoisomers of a plasmid preparation differ in linking number in increments of +/- 1. Thus, the observed linearization of each individual topoisomer following enzyme treatment, as a result of resolution of the crossovers associated with each topoisomer, showed that branch point migration to sensitive crossover positions must have occurred facilely. T7 endonuclease I randomly resolved across either axis of the cruciform, though some discrimination (related to the sequence specificity of the enzyme) was observed. The ten-nucleotide spacing between sensitive crossover positions is accounted for by an isomerization of the cruciform junction on branch point migration. An hypothesis is that this isomerization was imposed upon the cruciform junction by the change in helix twist (delta Tw) in the two branches that compose the topologically closed, circular domain of the plasmid. T7 endonuclease I may discriminate between the various isomeric forms and cleave a sensitive conformation that appears with every turn of branch migration which leads to the extrusion, or absorption, of two turns of helix from the circular core.
用噬菌体T7内切核酸酶I切割超螺旋质粒DNA后，对分支迁移十字形连接点在其中的可变位置进行了定位。T7内切核酸酶I特异性切割并由此解析了环状细菌质粒pSA1B.56A中十字形底部存在的霍利迪连接点。pSA1B.56A（含有由痘病毒端粒序列组成的322个碱基对的反向重复插入片段）中克隆序列的十字形挤出在体外使质粒底物发生拓扑松弛。因此，在克隆序列区域内鉴定出了许多交叉位置，反映了天然质粒制品中超螺旋密度的范围。在对质粒制品或单个拓扑异构体进行T7内切核酸酶I消化后，映射到病毒插入片段两条链上的内切核酸酶I敏感交叉位置通常相隔约十个核苷酸。每十个核苷酸出现一次敏感交叉对应于分支点迁移过程中质粒环状核心结构域中连接差（δLk）发生±2的变化。相比之下，质粒制品的单个拓扑异构体的连接数增量为±1。因此，酶处理后每个单独拓扑异构体的观察到的线性化，由于与每个拓扑异构体相关的交叉的解析，表明分支点向敏感交叉位置的迁移必定很容易发生。T7内切核酸酶I随机地在十字形的任一轴上进行解析，不过观察到了一些区分（与酶的序列特异性有关）。敏感交叉位置之间十个核苷酸的间距是由分支点迁移时十字形连接点的异构化造成的。一种假说是，这种异构化是由构成质粒拓扑封闭环状结构域的两个分支中螺旋扭曲（δTw）的变化施加在十字形连接点上的。T7内切核酸酶I可能会区分各种异构形式，并切割随着分支迁移每一圈出现的一种敏感构象，这会导致从环状核心挤出或吸收两圈螺旋。
