Structure and function of novel prokaryotic DNA transposases

我们对IS200/IS605转座酶家族代表成员的体外生化和结构研究表明，该家族使用一种完全新颖的重组途径，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特定位点的DNA结合域来识别其靶点：靶点识别是通过靶点与转座子DNA内部片段之间的碱基配对相互作用来完成的。这表明通过改变内部片段，靶向可以定向到新的目标部位的可能性。如果我们能做到这一点，这可能会允许将外源基因精确地引入染色体的良性位置或基因表达可以以细胞和发育特异性的方式适当控制的地方。

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.