Guzmán L M, Espinosa M
Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas Velázquez, Madrid, Spain.
J Mol Biol. 1997 Mar 7;266(4):688-702. doi: 10.1006/jmbi.1996.0824.
The streptococcal plasmid pMV158 replicates by the rolling circle mechanism. It encodes a relaxase protein of 494 residues, termed MobM, involved in conjugative mobilization. MobM protein was overproduced, purified, and shown specifically to relax supercoiled pMV158 DNA. The 5'-end and the 3'-end of the nick site introduced by MobM have been determined by sequencing and by primer extension analysis. The nucleophilic attack exerted by MobM is in the 5'-GpT-3' dinucleotide, within the sequence 5'-TAGTGTG/TTA-3'. Upon cleavage, MobM protein remains tightly associated with its target DNA, probably through a covalent bond. The pMV158 oriT did not exhibit homologies with known origins of transfer of plasmids from Gram-negative bacteria. However, several plasmids from Gram-positive hosts have a region identical or very similar to the pMV158 oriT. To our knowledge, this is the first demonstration of a relaxase activity of a mobilization protein from a plasmid replicating by the rolling circle mechanism.
链球菌质粒pMV158通过滚环机制进行复制。它编码一种由494个残基组成的松弛酶蛋白，称为MobM，参与接合转移。MobM蛋白被过量表达、纯化，并被证明能特异性地使超螺旋pMV158 DNA松弛。通过测序和引物延伸分析确定了由MobM引入的切口位点的5'端和3'端。MobM施加的亲核攻击发生在5'-GpT-3'二核苷酸处，位于5'-TAGTGTG/TTA-3'序列内。切割后，MobM蛋白可能通过共价键与其靶DNA紧密结合。pMV158 oriT与革兰氏阴性菌质粒的已知转移起始位点没有同源性。然而，来自革兰氏阳性宿主的几种质粒有一个与pMV158 oriT相同或非常相似的区域。据我们所知，这是首次证明通过滚环机制复制的质粒的转移蛋白具有松弛酶活性。
