Investigating the basis of substrate recognition in the pC221 relaxosome.
Investigating the basis of substrate recognition in the pC221 relaxosome.
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DOI：
10.1111/j.1365-2958.2006.05188.x
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发表时间：
2006-06
影响因子：
3.6
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中科院分区：
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转移起点（oriT）的切口是质粒DNA接合动员中必不可少的初始步骤。在葡萄球菌质粒pC 221的情况下，通过质粒特异性MobA松弛酶的切口是由DNA结合辅助蛋白MobC促进的;然而，MobC在此过程中的作用目前尚不清楚。在这项研究中，MobC结合的网站是通过DNA酶I足迹法确定的。MobC与oriT DNA在oriT nic位点上游的两个直接重复的9 bp序列mcb 1和mcb 2处相互作用，另外在mobC基因内的第三个简并重复序列mcb 3处相互作用。通过竞争性电泳迁移率变动测定间接地和通过表面等离子体共振研究直接地证实保守序列的结合活性。在mcb 2的突变废除可检测的切口活性，这表明该网站的MobC的结合是由MobA切口的先决条件。pC 221中每个结合位点的顺序定点诱变已经证明，所有三个都是动员所需的。MobA松弛酶，而不能单独结合到oriT DNA，被发现与MobC-oriT复合物和改变的MobC之间的区域mcb 2和nic网站的结合配置文件。oriT在该区域的突变定义了一个7 bp的序列，sra，这是必要的由MobA切口。pC 221和相关质粒pC 223的sra之间的四个不同碱基的交换足以在MobA特异性切口测定中交换它们的底物身份。基于这些观察，我们提出了一个模型的分层特异性组装的pC 221-家庭松弛体，从而一个共同的MobC：mcb复合物提出了oriT基板，然后只由同源MobA切口。
The nicking of the origin of transfer (oriT) is an essential initial step in the conjugative mobilization of plasmid DNA. In the case of staphylococcal plasmid pC221, nicking by the plasmid-specific MobA relaxase is facilitated by the DNA-binding accessory protein MobC; however, the role of MobC in this process is currently unknown. In this study, the site of MobC binding was determined by DNase I footprinting. MobC interacts with oriT DNA at two directly repeated 9 bp sequences, mcb1 and mcb2, upstream of the oriT nic site, and additionally at a third, degenerate repeat within the mobC gene, mcb3. The binding activity of the conserved sequences was confirmed indirectly by competitive electrophoretic mobility shift assays and directly by Surface Plasmon Resonance studies. Mutation at mcb2 abolished detectable nicking activity, suggesting that binding of this site by MobC is a prerequisite for nicking by MobA. Sequential site-directed mutagenesis of each binding site in pC221 has demonstrated that all three are required for mobilization. The MobA relaxase, while unable to bind to oriT DNA alone, was found to associate with a MobC–oriT complex and alter the MobC binding profile in a region between mcb2 and the nic site. Mutagenesis of oriT in this region defines a 7 bp sequence, sra, which was essential for nicking by MobA. Exchange of four divergent bases between the sra of pC221 and the related plasmid pC223 was sufficient to swap their substrate identity in a MobA-specific nicking assay. Based on these observations we propose a model of layered specificity in the assembly of pC221-family relaxosomes, whereby a common MobC:mcb complex presents the oriT substrate, which is then nicked only by the cognate MobA.