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，我们一直在继续探索IS 200/IS 605转座的机制。特别是，我们一直在研究转座子末端和识别DNA发夹之间的核苷酸数量（“接头长度”）如何影响IS 608转座，以及拟议的结构变化如何驱动从DNA链切割到链转移的过程。我们的数据与我们先前提出的旋转模型一致，其中两个灵活的α-螺旋相对于酶活性位点交替其构型，并且这些构型之间的来回-沿着“重置”步骤-驱动转座反应向前。 我们还一直在研究与细菌重复基因外回文序列（或REP）相关的推定转座酶。REP形成核苷酸茎环结构，并在许多细菌物种中大量分散。它们的绝对数量表明有一个过程导致它们在宿主物种中的扩增，并且已经提出这可能涉及与IS 200/IS 605转座酶密切相关的蛋白质。为了证实这一点，我们确定了来自E.大肠杆菌菌株MG 1655与DNA回文复合。事实上，它类似于IS 200/IS 605转座酶，并具有能够切割含有REP序列的某些DNA结构的特性。因此，它似乎可能是负责整个细菌基因组的REP序列的增殖，并已成为基因组进化的重要贡献者。 Curcio，M.J.和Derbyshire，K.M.（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 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.