de la Campa A G, del Solar G H, Espinosa M
Centro de Investigaciones Biológicas, C.S.I.C., Madrid, Spain.
J Mol Biol. 1990 May 20;213(2):247-62. doi: 10.1016/S0022-2836(05)80188-3.
The broad host range streptococcal plasmid pLS1 encodes the 24.2 kDa protein RepB, which is involved in the initiation of plasmid replication by an asymmetric rolling circle. RepB was overproduced in an Escherichia coli expression system and the protein was purified and characterized. Determination of the amino-terminal sequence of RepB protein showed that translation starts from the first AUG codon, which is preceded by an atypical ribosome-binding site sequence. RepB protein has in vitro-specific endonuclease and topoisomerase-like activities on the plasmid ori(+). Footprinting experiments showed that RepB protein binds to a DNA region that includes three direct repeats of 11 base-pairs. Initiation of replication of pLS1 could start by a RepB-generated specific nick introduced on the plasmid coding strand. However, as a striking difference with other Gram-positive replicons, the nick generated by RepB lies 86 base-pairs upstream from its binding region. To explain the action of RepB at a distance, complex structures of the pLS1 ori(+) are proposed.
广宿主范围的链球菌质粒pLS1编码24.2 kDa的蛋白质RepB，它通过不对称滚环参与质粒复制的起始。RepB在大肠杆菌表达系统中过量表达，并对该蛋白质进行了纯化和表征。RepB蛋白氨基末端序列的测定表明，翻译从第一个AUG密码子开始，该密码子之前是一个非典型的核糖体结合位点序列。RepB蛋白在质粒ori(+)上具有体外特异性核酸内切酶和拓扑异构酶样活性。足迹实验表明，RepB蛋白与一个包含三个11个碱基对直接重复序列的DNA区域结合。pLS1复制的起始可能由RepB在质粒编码链上产生的特异性切口引发。然而，与其他革兰氏阳性复制子的一个显著差异是，RepB产生的切口位于其结合区域上游86个碱基对处。为了解释RepB在远距离的作用，提出了pLS1 ori(+)的复杂结构。
