Site-specific recombinase and integrase activities of a conjugative relaxase in recipient cells
Site-specific recombinase and integrase activities of a conjugative relaxase in recipient cells
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DOI：
10.1073/pnas.0506081102
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发表时间：
2005-11-08
影响因子：
11.1
通讯作者：
Llosa, M
中科院分区：
文献类型：
--
作者：
Draper, O;César, CE;Llosa, M
结合松弛蛋白是通过对转移的DNA链进行位点特异性切割来启动细菌结合的蛋白质。在体外，它们在单链DNA上显示出链转移酶活性，这表明它们也可能负责转移DNA的再循环。在这项工作中，我们发现TrwC，质粒R388的松弛酶，在受体细胞中完全起作用，正如R388 TrwC突变体在受体细胞中的互补所显示的那样。在没有DNA转移的情况下也观察到TrwC转运到受体，尽管它仍然需要共轭偶联蛋白。除了偶联作用外，TrwC还能催化两个转移源（oriT）拷贝之间的位点特异性重组。破坏TrwC DNA链转移酶活性的突变也破坏了基因特异性重组。当trwc引导的DNA被接合地转移到受体细胞中时，含有两个oriT副本的质粒将经历重组。最后，我们展示了trwc依赖性的转移DNA整合到受体细胞的驻留或拷贝中。我们的研究结果表明，共轭松弛在受体细胞中是活跃的，在那里它执行缺口和链转移反应，这将需要再循环转移的DNA。这种TrwC在受体细胞中的位点特异性整合活性可能会导致未来的生物技术应用。
Conjugative relaxases are the proteins that initiate bacterial conjugation by a site-specific cleavage of the transferred DNA strand. In vitro, they show strand-transferase activity on single-stranded DNA, which suggests they may also be responsible for recircularization of the transferred DNA. In this work, we show that TrwC, the relaxase of plasmid R388, is fully functional in the recipient cell, as shown by complementation of an R388 trwC mutant in the recipient. TrwC transport to the recipient is also observed in the absence of DNA transfer, although it still requires the conjugative coupling protein. In addition to its role in conjugation, TrwC is able to catalyze site-specific recombination between two origin of transfer (oriT) copies. Mutations that abolish TrwC DNA strand-transferase activity also abolish oriT-specific recombination. A plasmid containing two oriT copies resident in the recipient cell undergoes recombination when a TrwC-piloted DNA is conjugatively transferred into it. Finally, we show TrwC-dependent integration of the transferred DNA into a resident oriT copy in the recipient cell. Our results indicate that a conjugative relaxase is active once in the recipient cell, where it performs the nicking and strand-transfer reactions that would be required to recircularize the transferred DNA. This TrwC site-specific integration activity in recipient cells may lead to future biotechnological applications.