Liao Shiping, Mao Chengde, Birktoft Jens J, Shuman Stewart, Seeman Nadrian C
Department of Chemistry, New York University, New York, New York 10003, USA.
Biochemistry. 2004 Feb 17;43(6):1520-31. doi: 10.1021/bi0358061.
Holliday junctions are intermediates in genetic recombination. They consist of four strands of DNA that flank a branch point. In natural systems, their sequences have 2-fold (homologous) sequence symmetry. This symmetry enables the molecules to undergo an isomerization, known as branch migration, that relocates the site of the branch point. Branch migration leads to polydispersity, which makes it difficult to characterize the physical properties of the junction and the effects of the sequence context flanking the branch point. Previous studies have reported two symmetric junctions that do not branch migrate: one that is immobilized by coupling to an asymmetric junction in a double crossover context, and a second that is based on molecules containing 5',5' and 3',3' linkages. Both are flawed by distorting the structure of the symmetric junction from its natural conformation. Here, we report an undistorted symmetric immobile junction based on the use of DNA parallelogram structures. We have used a series of these junctions to characterize the junction resolution reaction catalyzed by vaccinia virus DNA topoisomerase. The resolution reaction entails cleavage and rejoining at CCCTT/N recognition sites arrayed on opposing sides of the four-arm junction. We find that resolution is optimal when the scissile phosphodiester (Tp/N) is located two nucleotides 5' to the branch point on the helical strand. Covalent topoisomerase-DNA adducts are precursors to recombinant strands in all reactions, as expected. Kinetic analysis suggests a rate limiting step after the first-strand cleavage.
霍利迪连接体是基因重组过程中的中间体。它们由位于分支点两侧的四条DNA链组成。在自然系统中，它们的序列具有二重（同源）序列对称性。这种对称性使分子能够发生一种异构化，即分支迁移，从而重新定位分支点的位置。分支迁移导致多分散性，这使得难以表征连接体的物理性质以及分支点侧翼序列环境的影响。先前的研究报道了两种不发生分支迁移的对称连接体：一种是在双交换背景下通过与不对称连接体偶联而固定化的，另一种是基于含有5',5'和3',3'连接的分子。两者都存在缺陷，因为它们使对称连接体的结构偏离了其自然构象。在这里，我们报道了一种基于DNA平行四边形结构的未扭曲对称固定连接体。我们使用了一系列这些连接体来表征由痘苗病毒DNA拓扑异构酶催化的连接体拆分反应。拆分反应需要在排列在四臂连接体相对两侧的CCCTT/N识别位点处进行切割和重新连接。我们发现，当可切割的磷酸二酯（Tp/N）位于螺旋链上分支点5'端的两个核苷酸处时，拆分效果最佳。正如预期的那样，在所有反应中，共价拓扑异构酶-DNA加合物都是重组链的前体。动力学分析表明，在第一链切割后存在一个限速步骤。
