Dickie P, McFadden G, Morgan A R
Department of Biochemistry, University of Alberta, Edmonton, Canada.
J Biol Chem. 1987 Oct 25;262(30):14826-36.
Various branched DNA structures were created from synthetic, partly complementary oligonucleotides combined under annealing conditions. Appropriate mixtures of oligonucleotides generated three specific branched duplex DNA molecules: (i) a Holliday junction analog having a fixed (immobile) crossover bounded by four duplex DNA branches, (ii) a similar Holliday junction analog which is capable of limited branch migration and, (iii) a Y-junction, with three duplex branches and fixed branch point. Each of these novel structures was specifically cleaved by bacteriophage T7 gene 3 product, endonuclease I. The cleavage reaction "resolved" the two Holliday structure analogs into pairs of duplex DNA products half the size of the original molecules. The point of cleavage in the fixed-junction molecules was predominantly one nucleotide removed to the 5' side of the expected crossover position. Multiple cleavage positions were mapped on the Holliday junction with the mobile, or variable, branch point, to sites consistent with the unrestricted movement of the phosphodiester crossover within the region of limited dyad symmetry which characterizes this molecule. Based on the cleavage pattern observed with this latter substrate, the enzyme displayed a modest degree of sequence specificity, preferring a pyrimidine on the 3' side of the cleavage site. Branched molecules that were partial duplexes (lower order complexes which possessed single-stranded as well as duplex DNA branches) were also substrates for the enzyme. In these molecules, the cleaved phosphodiester bonds were in duplex regions only and predominantly one nucleotide to the 5' side of the branch point. The phosphodiester positions 5' of the branch point in single-stranded arms were not cleaved. Under identical reaction conditions, individually treated oligonucleotides were completely refractory. Thus, cleavage by T7 endonuclease I displays great structural specificity with an efficiency that can vary slightly according to the DNA sequence.
通过在退火条件下将合成的部分互补寡核苷酸组合，构建了各种分支DNA结构。合适的寡核苷酸混合物产生了三种特定的分支双链DNA分子：（i）一种霍利迪连接体类似物，其具有由四个双链DNA分支界定的固定（不可移动）交叉点；（ii）一种类似的霍利迪连接体类似物，其能够进行有限的分支迁移；以及（iii）一个Y型连接点，具有三个双链分支和固定的分支点。这些新型结构中的每一种都被噬菌体T7基因3产物内切酶I特异性切割。切割反应将两种霍利迪结构类似物“分解”为双链DNA产物对，其大小为原始分子的一半。固定连接分子中的切割点主要在预期交叉位置的5'侧去除一个核苷酸。多个切割位置被定位在具有可移动或可变分支点的霍利迪连接体上，这些位置与磷酸二酯交叉在表征该分子的有限二元对称区域内的无限制移动一致。基于用后一种底物观察到的切割模式，该酶表现出适度的序列特异性，在切割位点的3'侧更倾向于嘧啶。部分双链的分支分子（具有单链以及双链DNA分支的低阶复合物）也是该酶的底物。在这些分子中，切割的磷酸二酯键仅在双链区域，并且主要在分支点的5'侧一个核苷酸处。单链臂中分支点5'侧的磷酸二酯位置未被切割。在相同的反应条件下，单独处理的寡核苷酸完全不发生反应。因此，T7内切酶I的切割表现出很大的结构特异性，其效率会根据DNA序列略有变化。
