Structure and function of novel prokaryotic DNA transposases

我们一直在研究多重耐药细菌的插入序列(IS)运动，重点是产生碳青霉烯酶的肠杆菌科(CPE)。我们先前已经分析了来自NIH临床中心的几株临床和监测CPE分离株的ISS基因组环境，以靶点复制(TSD)及其分布模式为指导，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)。我们对通过IS26编码的转座酶进行DNA重排的机制很感兴趣，目前正在利用重组表达的蛋白质结合各种生化和生物物理分析来研究其性质。 从我们对来自NIH临床中心的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-插入”机制转座的插入序列所起的中心作用(1，2)。证明这一途径的重要性的是我们发现了ISApl1，一个复制-粘贴-插入转座子，如何通过形成一个简单重复的复合转座子来促进粘菌素抗性的出现(3)。尽管这一过程至关重要，但目前还没有关于这一过程如何运作的机械性信息。最近，我们已经从IS256家族中鉴定出一种通过该途径动员的可溶性的功能性转座酶，并且我们已经能够解析这种ISCth4转座酶与各种DNA底物的复合X射线结构。结果表明，它形成了一个不对称的二聚体转座体，其中N-末端结构域与转座子的一端结合，同时催化结构域移动以适应其不同的底物。这种不对称排列解释了转座子末端不寻常的不对称切割，这是复制-粘贴-插入转座的标志。 1.He，Hickman，Varani，Siguier，Chandler，Dekker，and Dyda(2015)插入序列IS26通过复制转座在临床分离的多重耐药细菌中重组质粒。MBio 3，e00762-15。 2.他，Chandler，Varani，Hickman，Dekker，and Dyda(2016)高后果耐药质粒的进化机制。MBio 6，e01987。 3.Snerrud，He，Chandler，Dekker，Hickman，McGann，and Dyda(2016)ISApl1转座粘菌素抗性基因mcr-1的模型。抗微生物药。化学其他探员。606973。

We have been investigating Insertion Sequence (IS) movements in multidrug resistant bacteria with a focus on carbapenemase-producing Enterobacteriaceae (CPE). We have previously analyzed the genomic contexts of ISs 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). Demonstrating the importance of this pathway was our discovery of how ISApl1, a copy-out-paste-in transposon, facilitated the emerge of colistin resistance by forming a tandemly repeated composite transposon (3). 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 solve X-ray structures of this ISCth4 transposase complexed with various DNA substrates. The results show that it forms an unusual asymmetric dimeric transpososome in which an array of N-terminal domains binds a single transposon end whilst the catalytic domain moves to accommodate its varying substrates. This asymmetric arrangement explains the unusual asymmetric cleavage of the transposon ends that is a hallmark of copy-out-paste-in 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. 3. Snesrud, He, Chandler, Dekker, Hickman, McGann, and Dyda (2016) A model for transposition of the colistin resistance gene mcr-1 by ISApl1. Antimicrob. Agents Chemother. 60, 6973.