Department of Microbiology, Cornell University, Ithaca, NY 14853, USA.
Mol Microbiol. 2011 Sep;81(6):1593-606. doi: 10.1111/j.1365-2958.2011.07789.x. Epub 2011 Aug 23.
Vegetative replication and partitioning of many plasmids and some chromosomes of alphaproteobacteria are directed by their repABC operons. RepA and RepB proteins direct the partitioning of replicons to daughter cells, while RepC proteins are replication initiators, although they do not resemble any characterized replication initiation protein. Here we show that the replication origin of an Agrobacterium tumefaciens Ti plasmid resides fully within its repC gene. Purified RepC bound to a site within repC with moderate affinity, high specificity and with twofold cooperativity. The binding site was localized to an AT-rich region that contains a large number of GANTC sites, which have been implicated in replication regulation in related organisms. A fragment of RepC containing residues 26-158 was sufficient to bind DNA, although with limited sequence specificity. This portion of RepC is predicted to have structural homology to members of the MarR family of transcription factors. Overexpression of RepC in A. tumefaciens caused large increases in copy number in cis but did not change the copy number of plasmids containing the same oriV sequence in trans, confirming other observations that RepC functions only in cis.
许多α变形菌的质粒和一些染色体的植物寄生菌通过 repABC 操纵子进行营养复制和分配。RepA 和 RepB 蛋白指导复制子分配到子细胞，而 RepC 蛋白是复制起始蛋白，尽管它们与任何已鉴定的复制起始蛋白都不相似。在这里，我们表明根癌土壤杆菌 Ti 质粒的复制起点完全位于其 repC 基因内。纯化的 RepC 与 repC 内的一个位点结合，具有中等亲和力、高特异性和两倍协同性。该结合位点定位于富含 AT 的区域，该区域包含大量 GANTC 位点，这些位点与相关生物体内的复制调控有关。含有残基 26-158 的 RepC 片段足以结合 DNA，尽管具有有限的序列特异性。该 RepC 部分预测具有与 MarR 家族转录因子成员的结构同源性。根癌土壤杆菌中 RepC 的过表达导致顺式中拷贝数的大幅增加，但不会改变反式中含有相同 oriV 序列的质粒的拷贝数，这证实了 RepC 仅在顺式中起作用的其他观察结果。
