Type IA topoisomerases interact with G-strand and T-strand ssDNA to regulate DNA topology. However, simultaneous binding of two ssDNA segments to a type IA topoisomerase has not been observed previously. We report here the crystal structure of a type IA topoisomerase with ssDNA segments bound in opposite polarity to the N- and C-terminal domains. Titration of small ssDNA oligonucleotides to Mycobacterium smegmatis topoisomerase I with progressive C-terminal deletions showed that the C-terminal region has higher affinity for ssDNA than the N-terminal active site. This allows the C-terminal domains to capture one strand of underwound negatively supercoiled DNA substrate first and position the N-terminal domains to bind and cleave the opposite strand in the relaxation reaction. Efficiency of negative supercoiling relaxation increases with the number of domains that bind ssDNA primarily with conserved aromatic residues and possibly with assistance from polar/basic residues. A comparison of bacterial topoisomerase I structures showed that a conserved transesterification unit (N-terminal toroid structure) for cutting and rejoining of a ssDNA strand can be combined with two different types of C-terminal ssDNA binding domains to form diverse bacterial topoisomerase I enzymes that are highly efficient in their physiological role of preventing excess negative supercoiling in the genome.

中文翻译：

---

来自耻垢分枝杆菌拓扑异构酶 I 结构的机制见解，其中 ssDNA 结合到 N 端和 C 端结构域。

IA 型拓扑异构酶与 G 链和 T 链 ssDNA 相互作用，调节 DNA 拓扑结构。然而，之前尚未观察到两个 ssDNA 片段同时与 IA 型拓扑异构酶结合。我们在此报告了 IA 型拓扑异构酶的晶体结构，其 ssDNA 片段以相反极性结合到 N 端和 C 端结构域。将小 ssDNA 寡核苷酸滴定到具有渐进 C 端缺失的耻垢分枝杆菌拓扑异构酶 I 上，结果表明 C 端区域对 ssDNA 的亲和力高于 N 端活性位点。这使得 C 端结构域首先捕获缠绕下的负超螺旋 DNA 底物的一条链，并定位 N 端结构域以在弛豫反应中结合和切割相反的链。负超螺旋弛豫的效率随着主要与保守芳香残基结合 ssDNA 的结构域数量的增加而增加，并且可能在极性/碱性残基的帮助下。细菌拓扑异构酶 I 结构的比较表明，用于切割和重新连接 ssDNA 链的保守酯交换单元（N 端环形结构）可以与两种不同类型的 C 端 ssDNA 结合域组合，形成多种细菌拓扑异构酶 I 酶，在防止基因组中过度负超螺旋的生理作用方面非常有效。