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 continuing to explore the mechanism of IS200/IS605 transposition. In particular, we have been investigating how the number of nucleotides between the transposon ends and the recognition DNA hairpin (the "linker length") affects IS608 transposition, and also how a proposed structural change drives the process from DNA strand cleavage to strand transfer. Our data is consistent with our previously proposed rotation model in which two flexible alpha-helices alternate their configuration with respect to the enzyme active sites, and that the back-and-forth between these configurations - along with a "reset" step - drives the transposition reaction forward. We have also been studying the putative transposase associated with bacterial Repeated Extragenic Palindromic Sequences (or REPs). REPs form nucleotide stem-loop structures and are found scattered in high numbers in many bacterial species. Their sheer number suggests there was a process that led to their expansion in their host species, and it has been proposed that this might involve an protein closely related to the IS200/IS605 transposases. To confirm this, we determined the structure of the TnpA(REP) from E. coli strain MG1655 in complex with a DNA palindrome. Indeed, it resembles the IS200/IS605 transposases and shares the property of being able to cleave certain DNA structures that contain REP sequences. Thus, it appears likely that it has been responsible for the proliferation of REP sequences throughout bacterial genomes, and has been an important contributor to genome evolution. 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.