Structure and function of novel prokaryotic DNA transposases

我们一直在研究多重耐药菌中的插入序列（IS）移动，重点是产碳青霉烯酶的肠杆菌科（CPE）。一个插入序列IS 26经常与耐药决定簇相关，但其作用仍不清楚。我们之前已经分析了NIH临床中心的几种临床和监测CPE分离株中70个IS 26拷贝的基因组背景，使用靶位点重复（TSD）及其分布模式作为指导，发现大部分质粒重组是由IS 26分子内复制易位引起的，包括复制子融合、DNA倒位和缺失（1，2）。我们有兴趣了解这些DNA重排背后的IS 26编码的转座酶的机制，目前正在研究其特性，使用重组表达的蛋白质结合各种生化和生物物理测定。 从我们对NIH临床中心CPE分离株的分析中出现的另一个结果是认识到使用所谓的“复制-输出-粘贴-输入”机制转座的插入序列所起的中心作用（1，2）。尽管它的核心重要性，有没有目前的机械信息，关于这个过程是如何工作的。最近，我们已经确定了一个可溶性和功能性的转座酶从IS 256家庭，这是动员通过这一途径，我们已经能够获得衍射晶体的转座酶与各种DNA底物的复合物。我们目前正在分析转座酶与转座子末端DNA结合的结果结构，以了解它如何协调这种复制性DNA转座途径。 1. He，Hickman，Varani，Siguier，钱德勒德勒，Dekker，and Dyda（2015）Insertion Sequence IS26 reorganizes plasmid in clinically isolated multidrug resistant bacteria by replicative translocation. mBio 3，e00762-15。 2.他，钱德勒，瓦拉尼，希克曼，德克尔和Dyda（2016）进化机制在高后果耐药质粒。mBio 6，e01987。

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.