A new domain of conjugative relaxase TrwC responsible for efficient oriT-specific recombination on minimal target sequences
A new domain of conjugative relaxase TrwC responsible for efficient oriT-specific recombination on minimal target sequences
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DOI：
10.1111/j.1365-2958.2006.05437.x
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发表时间：
2006-11-01
影响因子：
3.6
通讯作者：
Llosa, Matxalen
中科院分区：
文献类型：
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作者：
Cesar, Carolina Elvira;Machon, Cristina;Llosa, Matxalen
我们发现，松弛酶TrwC促进两个直接重复的oriTs之间的重组，而相关的松弛酶TraI的F和pKM 101不。有效的重组也需要松弛体辅助蛋白TrwA，即使在OriT的TrwA结合位点删除后，表明TrwA的效果是由蛋白质-蛋白质相互作用介导的。TrwC松弛酶结构域是必需的，但不足以有效地催化重组。用TrwC的N-末端600个残基获得完全重组酶活性。重组所需的最小靶序列在两个所涉及的oriT中的每一个上都是不同的：oriT 1可以减少到nic位点和TrwC结合位点，而oriT 2需要一个扩展的序列，包括一组不需要缀合的干扰子。TrwC介导的转移DNA整合到一个居民oriT拷贝需要一个完整的oriT在收件人。我们观察到串联oriTs之间的重组效率的显着变化连接到质粒复制和转录的方向，通过oriT 1。我们提出，重组是由在oriT 1产生的单链DNA，导致TrwC切口触发。所得的TrwC-DNA复合物与oriT 2反应，切除插入的DNA。这个中间体可以通过宿主编码的复制函数来解析。
We show that relaxase TrwC promotes recombination between two directly repeated oriTs while related relaxases TraI of F and pKM101 do not. Efficient recombination required also relaxosome accessory protein TrwA even after deletion of TrwA binding sites at oriT, suggesting that the effect of TrwA is mediated by protein-protein interactions. TrwC relaxase domain was necessary but not sufficient to catalyse recombination efficiently. Full recombinase activity was obtained with the N-terminal 600 residues of TrwC. The minimal target sequences required for recombination were different at each of the two involved oriTs: oriT1 could be reduced to the nic site and TrwC binding site, while oriT2 required an extended sequence including a set of iterons that are not required for conjugation. TrwC-mediated integration of a transferred DNA into a resident oriT copy required a complete oriT in the recipient. We observed dramatic changes in the efficiency of recombination between tandem oriTs linked to the direction of plasmid replication and transcription through oriT1. We propose that recombination is triggered by the generation of a single-stranded DNA at oriT1 that causes TrwC nicking. The resulting TrwC-DNA complex reacts with oriT2, excising the intervening DNA. This intermediate can be resolved by host-encoded replication functions.