Bhattacharjee M K, Meyer R J
Department of Microbiology, University of Texas at Austin 78712.
Nucleic Acids Res. 1993 Sep 25;21(19):4563-8. doi: 10.1093/nar/21.19.4563.
MobA protein, encoded by the broad host-range plasmid R1162, is required for conjugal mobilization of this plasmid. The protein is an essential part of the relaxosome, and is also necessary for the termination of strand transfer. In vitro, MobA is a nuclease specific for one of the two DNA strands of the origin of transfer (oriT). The protein can cleave this strand at the same site that is nicked in the relaxosome, and can also ligate the DNA. We show here that purified MobA protein forms a complex that is specific for this single oriT strand. The complex is unusually stable, with a half-life of approximately 95 min, is not disrupted by hybridization with the complementary strand, and reforms rapidly after boiling. Both the inverted repeat within oriT, and the eight bases between this repeat and the site cleaved by MobA, are required for binding by the protein. Mutations reducing base complementarity between the arms of the inverted repeat also decrease binding. This effect is partially suppressed by second-site mutations restoring complementarity. These results parallel the effects of these mutations on termination. Footprinting experiments with P1 nuclease indicate that the DNA between the inverted repeat and the nick site is protected by MobA, but that pairing between the arms of the repeat, which occurs in the absence of protein, is partially disrupted. Our results suggest that termination of strand transfer during conjugation involves tight binding of the MobA protein to the inverted repeat and adjacent oriT DNA. This complex positions the protein for ligation of the ends of the transferred strand, to reform a circular plasmid molecule.
MobA蛋白由广宿主范围质粒R1162编码，是该质粒接合转移所必需的。该蛋白是松弛体的重要组成部分，也是链转移终止所必需的。在体外，MobA是一种对转移起始点（oriT）两条DNA链之一具有特异性的核酸酶。该蛋白可在松弛体中被切口的同一位置切割这条链，并且还能连接DNA。我们在此表明，纯化的MobA蛋白形成了一种对这条单一oriT链具有特异性的复合物。该复合物异常稳定，半衰期约为95分钟，不会因与互补链杂交而被破坏，并且在煮沸后能迅速重新形成。oriT内的反向重复序列以及该重复序列与MobA切割位点之间的八个碱基，都是蛋白结合所必需的。降低反向重复序列臂之间碱基互补性的突变也会减少结合。恢复互补性的第二位点突变可部分抑制这种效应。这些结果与这些突变对终止的影响相似。用P1核酸酶进行的足迹实验表明，反向重复序列与切口位点之间的DNA受到MobA的保护，但在没有蛋白的情况下重复序列臂之间发生的配对被部分破坏。我们的结果表明，接合过程中链转移的终止涉及MobA蛋白与反向重复序列及相邻oriT DNA的紧密结合。这种复合物使蛋白定位以连接转移链的末端，从而重新形成环状质粒分子。
