Laboratoire de Microbiologie et Génétique Moléculaires, Centre National de Recherche Scientifique, Unité Mixte de Recherche 5100, 118 Rte de Narbonne, F31062 Toulouse Cedex, France.
Nucleic Acids Res. 2011 Oct;39(19):8503-12. doi: 10.1093/nar/gkr566. Epub 2011 Jul 10.
Single-stranded (ss) transposition, a recently identified mechanism adopted by members of the widespread IS200/IS605 family of insertion sequences (IS), is catalysed by the transposase, TnpA. The transposase of IS608, recognizes subterminal imperfect palindromes (IP) at both IS ends and cleaves at sites located at some distance. The cleavage sites, C, are not recognized directly by the protein but by short sequences 5' to the foot of each IP, guide (G) sequences, using a network of canonical ('Watson-Crick') base interactions. In addition a set of non-canonical base interactions similar to those found in RNA structures are also involved. We have reconstituted a biologically relevant complex, the transpososome, including both left and right ends and TnpA, which catalyses excision of a ss DNA circle intermediate. We provide a detailed picture of the way in which the IS608 transpososome is assembled and demonstrate that both C and G sequences are essential for forming a robust transpososome detectable by EMSA. We also address several questions central to the organization and function of the ss transpososome and demonstrate the essential role of non-canonical base interactions in the IS608 ends for its stability by using point mutations which destroy individual non-canonical base interactions.
单链转位是一种最近被广泛的 IS200/IS605 家族插入序列（IS）成员采用的机制，由转座酶 TnpA 催化。IS608 的转座酶识别两端的亚末端不完全回文（IP），并在距离一定的位置切割。切割位点 C 不是由蛋白质直接识别，而是由每个 IP 脚的 5'端的短序列（引导序列 G）识别，使用标准的（沃森-克里克）碱基相互作用网络。此外，还涉及到一组类似于 RNA 结构中发现的非标准碱基相互作用。我们重新构建了一个具有生物学相关性的复合物，即转座体，包括左、右末端和 TnpA，它催化 ssDNA 环中间体的切除。我们提供了一个详细的图片，展示了 IS608 转座体的组装方式，并证明了 C 和 G 序列对于形成稳定的可通过 EMSA 检测的转座体都是必不可少的。我们还解决了与 ss 转座体的组织和功能有关的几个核心问题，并通过使用破坏单个非标准碱基相互作用的点突变，证明了非标准碱基相互作用对 IS608 末端稳定性的重要作用。
