Structure and function of novel prokaryotic DNA transposases

我们对IS 200/IS 605转座酶家族的代表性成员进行的体外生物化学和结构研究表明，该家族使用了一种全新的重组途径，仅涉及单链DNA的运动。一个特别令人惊讶的发现是转座酶通过DNA-DNA相互作用而不是使用位点特异性DNA结合结构域识别其靶位点：靶位点识别是通过靶位点和转座子DNA的内部片段之间的碱基配对相互作用完成的。这表明，通过改变内部片段，靶向可以指向新的靶位点。如果我们能做到这一点，这可能允许将外源基因精确引入染色体中的良性位置，或者基因表达可以以细胞和发育特异性方式适当控制的位置。 在我们最近的工作中，我们一直在探索IS 200/IS 605转座的两个方面。第一个是针对转座如何在细胞中发生的有趣问题，因为转座子及其靶必须是单链的，但细胞中的大多数DNA是双链的。我们已经使用了各种遗传和体内实验，以表明IS 608转座与复制叉处滞后链上ssDNA的可用性密切相关。 我们一直在研究的另一个方面是通过将我们的研究扩展到IS 200超家族的另一个成员ISDra 2来确定IS 608转座的特征是否可推广到整个超家族。ISDra 2是特别令人感兴趣的，因为其转座在紫外线或γ辐射后在耐辐射异常球菌中特异性诱导（Mennecier et al.，2006年）。我们已经解决了一系列的ISDra 2转座酶-DNA复合物，并表明，虽然许多针对IS 608发现的靶向原理是适用的，但存在机械上的重要差异。一个值得注意的区别是ISDra 2在其靶位点识别五个核苷酸，而不是IS 608的四个。ISDra 2实现这一点的方式扩展了我们对转座机制的理解。我们还能够在结构上捕获沿着该途径的预裂解状态，这是我们使用IS 608系统所无法获得的快照。 Curcio，M.J.和Derbyshire，K.M.等人（2003）Nat. Rev. Mol. Cell. Biol.4，865-877. Debets-Ossenkopp，Y.J.，等人（1999）Antimicrob.探员Chemother 43，2657-2662。 Kersulyte，D.，等人（2002）J. Bacteriol. 184，992-1002。 Mennecier，S.，Servant，P.，Coste，G.，Bailone，A.，和Sommer，S.（2006）Mol. Microbiol. 59，317-325。 Sebaihia，M.等人（2006）Nature Genet. 38，779-786。

Our combined in vitro biochemical and structural studies on a representative member of the IS200/IS605 transposase family demonstrated that this family uses a completely novel recombination pathway involving the movement of only single-stranded DNA. One particularly surprising discovery was that the transposase recognizes its target site through DNA-DNA interactions rather than using a site-specific DNA binding domain: target site recognition is accomplished by base pairing interactions between the target site and an internal segment of transposon DNA. This suggests the possibility that by changing the internal segment, targeting could be directed to novel target sites. If we can do this, this might allow the precise introduction of exogenous genes into benign locations in chromosomes or places where gene expression can be appropriately controlled in a cell- and development-specific manner. In our recent work, we have been exploring two aspects of IS200/IS605 transposition. The first is directed towards the intriguing question of how transposition occurs in cells since both the transposon and its target must be single-stranded yet most DNA in cells is double-stranded. We have used a variety of genetic and in vivo experiments to show that IS608 transposition is closely linked to the availability of ssDNA on the lagging strand at the replication fork. The other aspect we have been investigating is to determine if features of IS608 transposition are generalizable to the entire superfamily, by expanding our studies to another member of the IS200 superfamily, ISDra2. ISDra2 is particularly interesting as its transposition is specifically induced in Deinococcus radiodurans upon UV or gamma irradiation (Mennecier et al., 2006). We have solved a series of ISDra2 transposase-DNA complexes, and shown that although many of the principles of targeting found for IS608 are applicable, there are mechanistically important differences. One noteworthy difference is that ISDra2 recognizes five nucleotides at its target site, rather than the four of IS608. The manner in which ISDra2 accomplishes this extends our understanding of the mechanism of transposition. We have also been able to structurally capture the pre-cleavage state along the pathway, a snapshot that eluded us with the IS608 system. Curcio, M.J. and Derbyshire, K.M. (2003) Nat. Rev. Mol. Cell. Biol. 4, 865-877. Debets-Ossenkopp, Y.J., et al. (1999) Antimicrob. Agents Chemother. 43, 2657-2662. Kersulyte, D., et al. (2002) J. Bacteriol. 184, 992-1002. Mennecier, S., Servant, P., Coste, G., Bailone, A., and Sommer, S. (2006) Mol. Microbiol. 59, 317-325. Sebaihia, M. et al. (2006) Nature Genet. 38, 779-786.