Structure and function of novel prokaryotic DNA transposases

我们一直在研究多药耐药细菌的插入序列（IS）运动，重点是产碳青霉烯酶的肠杆菌科（CPE）。其中一个插入序列IS26经常与抗性决定因子相关，但其作用尚不清楚。我们之前分析了来自NIH临床中心的几个临床和监测CPE分离株的70个IS26拷贝的基因组背景，使用目标位点复制（TSDs）及其分布模式作为指导，发现很大一部分质粒重组是由IS26分子内复制转位引起的，包括复制子融合、DNA倒置和缺失（1,2）。我们有兴趣了解由IS26编码的转座酶进行DNA重排的机制，目前正在利用重组表达蛋白结合各种生化和生物物理分析研究其特性。

We have been investigating Insertion Sequence (IS) movements in multidrug resistant bacteria with a focus on carbapenemase-producing Enterobacteriaceae (CPE). One Insertion Sequence, IS26, is frequently associated with resistance determinants, but its role remained unclear. We have previously analyzed the genomic contexts of 70 IS26 copies in several clinical and surveillance CPE isolates from the NIH Clinical Center, using target site duplications (TSDs) and their distribution patterns as guides, and discovered that a large fraction of plasmid reorganizations result from IS26 intramolecular replicative transpositions, including replicon fusions, DNA inversions, and deletions (1,2). We are interested in learning about the mechanisms behind these DNA rearrangements by the transposase encoded by IS26, and are currently studying its properties using recombinantly expressed protein in combination with various biochemical and biophysical assays. Another result that emerged from our analysis of CPE isolates from the NIH Clinical Center was the recognition of the central role played by Insertion Sequences that transpose using the so-call "copy-out-paste-in" mechanism (1,2). Despite its central importance, there is no current mechanistic information available regarding how this process works. Recently, we have identified a soluble and functional transposase from the IS256 family that is mobilized by this pathway, and we have been able to obtain diffracting crystals of the transposase complexed with various DNA substrates. We are currently analyzing the resulting structures of the transposase bound to transposon-end DNA to understand how it orchestrates this pathway of replicative DNA transposition. 1. He, Hickman, Varani, Siguier, Chandler, Dekker, and Dyda (2015) Insertion Sequence IS26 reorganizes plasmids in clinically isolated multidrug-resistant bacteria by replicative transposition. mBio 3, e00762-15. 2. He, Chandler, Varani, Hickman, Dekker, and Dyda (2016) Mechanism of evolution in high-consequence drug resistance plasmids. mBio 6, e01987.