Integron cassette insertion: a recombination process involving a folded single strand substrate
Integron cassette insertion: a recombination process involving a folded single strand substrate
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DOI：
10.1038/sj.emboj.7600898
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发表时间：
2005-12-21
期刊：
影响因子：
11.4
通讯作者：
Mazel, D
中科院分区：
文献类型：
--
作者：
Bouvier, M;Demarre, G;Mazel, D
整合子在革兰氏阴性病原体中抗生素耐药基因的传播中起主要作用。在整合子特异性整合酶IntI催化的反应中，整合子基因盒形成携带重组位点attC的环状中间体，并在第二位点attI插入整合子平台。IntI1整合子整合酶优先结合单链attC的“底链”。我们已经解决了插入机制，在体内使用重组试验，利用质粒缀合，专门提供不同的整合酶重组底物的顶部或底部链。单链attC位点与attI位点的重叠是一条链的1000倍。相反，在任一attI链的接合转移之后，与attC的重组是非常不利的。这些结果和使用假定的attC茎环内的突变获得的那些结果强烈支持一种新的整合子盒插入模型，其中单个底部attC链采用产生双链重组位点的折叠结构。因此，重组将插入单链盒，其必须随后加工。
Integrons play a major role in the dissemination of antibiotic resistance genes among Gram-negative pathogens. Integron gene cassettes form circular intermediates carrying a recombination site, attC, and insert into an integron platform at a second site, attI, in a reaction catalyzed by an integron-specific integrase IntI. The IntI1 integron integrase preferentially binds to the 'bottom strand' of single-stranded attC. We have addressed the insertion mechanism in vivo using a recombination assay exploiting plasmid conjugation to exclusively deliver either the top or bottom strand of different integrase recombination substrates. Recombination of a single-stranded attC site with an attI site was 1000-fold higher for one strand than for the other. Conversely, following conjugative transfer of either attI strand, recombination with attC is highly unfavorable. These results and those obtained using mutations within a putative attC stem-and-loop strongly support a novel integron cassette insertion model in which the single bottom attC strand adopts a folded structure generating a double strand recombination site. Thus, recombination would insert a single strand cassette, which must be subsequently processed.