Du S M, Wang H, Tse-Dinh Y C, Seeman N C
Department of Chemistry, New York University, New York 10003.
Biochemistry. 1995 Jan 17;34(2):673-82. doi: 10.1021/bi00002a035.
Two synthetic DNA molecules that can be knotted have been employed as substrates for E. coli DNA topoisomerases I and III. Both molecules contain 104 nucleotides, including sequences that can form two single-turn helical domains, connected by single-stranded oligo(dT) linkers in an X-Y-X'-Y' pairing motif. One of the knots can be ligated to form cyclic molecules with the topologies of a circle, a trefoil knot with negative nodes, or a figure-8 knot. Cyclic molecules constructed from the other molecule can form a circle, a figure-8 knot, and trefoil knots with either positive or negative nodes. The topologically negative nodes in these knots are derived from right-handed B-DNA, and the positive nodes are derived from left-handed Z-DNA. The topoisomerases can catalyze the interconversion of the different topological forms of these molecules, as a function of solution conditions and the extent to which they favor B-DNA or Z-DNA. The enzymes appear to catalyze a single strand-passage event at a time. The topoisomerases can catalyze strand passage events involving both positive and negative nodes as substrates. Gel retention experiments show that both knots can bind up to four molecules of E. coli DNA topoisomerase I. The thermal denaturation of the domains of a trefoil knot closely related to these knots suggests that the two helical domains are uncoupled, so the single-stranded linkers in the knots are not taut. Chemical ligation experiments yield a distribution of products similar to those of enzymatic ligation, showing that the ATP cofactor in DNA knot ligation does not appear to skew the products markedly. Knots that are stressed by being placed in unfavorable solution conditions have been shown to be a highly sensitive system for detecting topoisomerase activity.
两种能够形成纽结的合成DNA分子被用作大肠杆菌DNA拓扑异构酶I和III的底物。这两种分子均含有104个核苷酸，包括能够形成两个单链螺旋结构域的序列，这些结构域通过单链寡聚（dT）接头以X-Y-X'-Y'配对基序相连。其中一个纽结可以被连接形成具有环形、负节点三叶结或8字结拓扑结构的环状分子。由另一种分子构建的环状分子可以形成环形、8字结以及具有正节点或负节点的三叶结。这些纽结中拓扑学上的负节点源自右手性B-DNA，而正节点源自左手性Z-DNA。拓扑异构酶能够催化这些分子不同拓扑形式之间的相互转化，这是溶液条件以及它们对B-DNA或Z-DNA偏好程度的函数。这些酶似乎一次催化一个单链通过事件。拓扑异构酶能够催化涉及正节点和负节点作为底物的链通过事件。凝胶滞留实验表明，这两种纽结都能结合多达四个大肠杆菌DNA拓扑异构酶I分子。与这些纽结密切相关的三叶结结构域的热变性表明，两个螺旋结构域是解偶联的，因此纽结中的单链接头并不紧绷。化学连接实验产生的产物分布与酶促连接的产物相似，表明DNA纽结连接中的ATP辅因子似乎不会显著影响产物。已证明，置于不利溶液条件下而受到应力作用的纽结是检测拓扑异构酶活性的高度敏感系统。
