TOPOLOGICAL TRANSFORMATIONS OF SYNTHETIC DNA KNOTS
TOPOLOGICAL TRANSFORMATIONS OF SYNTHETIC DNA KNOTS
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DOI：
10.1021/bi00002a035
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发表时间：
1995-01-17
期刊：
影响因子：
2.9
通讯作者：
SEEMAN, NC
中科院分区：
文献类型：
--
作者：
DU, SM;WANG, H;SEEMAN, NC
两种可以打结的合成DNA分子已被用作E. coli DNA拓扑异构酶I和III。这两种分子都含有104个核苷酸，包括可以形成两个单圈螺旋结构域的序列，通过X-Y-X '-Y'配对基序中的单链寡核苷酸（dT）接头连接。其中一个结可以连接形成具有圆形拓扑结构的环状分子，具有负节点的三叶结或8字形结。由另一个分子构成的环状分子可以形成一个圆圈，一个8字形结，以及带有正节点或负节点的三叶结。这些结中的拓扑负节点来自右手B-DNA，正节点来自左手Z-DNA。拓扑异构酶可以催化这些分子的不同拓扑形式的相互转化，作为溶液条件和它们有利于B-DNA或Z-DNA的程度的函数。这些酶似乎一次催化一个单链通过事件。拓扑异构酶可以催化涉及作为底物的正节点和负节点的链通过事件。凝胶保留实验表明，这两个结可以结合多达四个分子的E。coli DNA拓扑异构酶I。与这些结密切相关的三叶结的结构域的热变性表明两个螺旋结构域是解偶联的，因此结中的单链接头不是拉紧的。化学连接实验产生的产物分布类似于酶促连接，表明在DNA结连接的ATP辅因子并没有出现显着偏斜的产品。通过放置在不利的溶液条件下而受到应力的结已被证明是用于检测拓扑异构酶活性的高度灵敏的系统。
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.