Llosa M, Bolland S, Grandoso G, de la Cruz F
Departamento de Biología Molecular, Universidad de Cantabria, Santander, Spain.
J Bacteriol. 1994 Jun;176(11):3210-7. doi: 10.1128/jb.176.11.3210-3217.1994.
Plasmids containing a direct repeat of plasmid R388 oriT are capable of site-specific recombination, which results in deletion of the intervening DNA. This reaction occurs in the presence, but not in the absence, of the region of R388 implicated in DNA processing during conjugation. This region contains three genes, trwA, trwB, and trwC. By using mutants of each of the three genes, it was demonstrated that only trwC is required for the oriT-specific recombination. Further analysis showed that the N-terminal 272 amino acids of the protein are sufficient to catalyze recombination. TrwC is also capable of promoting intermolecular recombination between two plasmids containing oriT, suggesting that double-strand breaks in both plasmid DNAs are involved in the process. Additionally, intramolecular recombination between R388 oriT and R46 oriT did not occur in the presence of both nickases. This suggests that the half-reactions at each oriT are not productive if they occur separately; therefore, an interaction between the recombination complexes formed at each recombining site is required. This is the first report in which a nicking-closing enzyme involved in conjugal DNA transfer promotes oriT-specific recombination of double-stranded DNA in the absence of conjugation.
含有质粒R388 oriT直接重复序列的质粒能够进行位点特异性重组，这会导致中间DNA的缺失。该反应在存在参与接合过程中DNA加工的R388区域时发生，但在不存在该区域时不发生。该区域包含三个基因，trwA、trwB和trwC。通过使用这三个基因各自的突变体，证明oriT特异性重组仅需要trwC。进一步分析表明，该蛋白质的N端272个氨基酸足以催化重组。TrwC还能够促进两个含有oriT的质粒之间的分子间重组，这表明两个质粒DNA中的双链断裂都参与了该过程。此外，在两种切口酶存在的情况下，R388 oriT和R46 oriT之间未发生分子内重组。这表明如果每个oriT处的半反应单独发生则没有效果；因此，在每个重组位点形成的重组复合物之间需要相互作用。这是第一份关于参与接合DNA转移的切口-封闭酶在无接合情况下促进双链DNA的oriT特异性重组的报告。
