Cha Kyung-Il, Lim Kihong, Jang Sehwan, Lim Wangjin, Kim Taehyung, Chang Hyoihl
School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea.
J Microbiol Biotechnol. 2007 Nov;17(11):1841-7.
We previously identified the origin of replication of p703/5, a small cryptic plasmid from the KBL703 strain of Enterococcus faecalis. The origin of replication contains putative regulatory cis-elements required for replication and a replication initiator (RepA) gene. The replicon of p703/5 is similar in its structural organization to theta-type plasmids, and RepA is homologous to a family of Rep proteins identified in several plasmids from Gram-positive bacteria. Here, we report molecular interactions between RepA and the replication origin of p703/5. DNase I footprinting using recombinant RepA together with electrophoretic mobility shift assays confirmed the binding of RepA to the replication origin of p703/5 via iterons and an inverted repeat. We also demonstrated the formation of RepA dimers and the different binding of RepA to the iteron and the inverted repeat using gel filtration chromatographic analysis, a chemical crosslinking assay, and electrophoretic mobility shift assays in the presence of guanidine hydrochloride. Our results suggest that RepA plays a regulatory role in the replication of the enterococcal plasmid p703/5 via mechanisms similar to those of typical iteron-carrying theta-type plasmids.
我们之前鉴定了p703/5的复制起点，p703/5是来自粪肠球菌KBL703菌株的一个小型隐蔽质粒。该复制起点包含复制所需的假定调控顺式元件和一个复制起始蛋白（RepA）基因。p703/5的复制子在结构组织上与θ型质粒相似，并且RepA与在革兰氏阳性菌的几种质粒中鉴定出的Rep蛋白家族同源。在此，我们报道了RepA与p703/5复制起点之间的分子相互作用。使用重组RepA进行的DNase I足迹实验以及电泳迁移率变动分析证实了RepA通过重复序列和一个反向重复序列与p703/5的复制起点结合。我们还通过凝胶过滤色谱分析、化学交联实验以及在盐酸胍存在下的电泳迁移率变动分析证明了RepA二聚体的形成以及RepA与重复序列和反向重复序列的不同结合。我们的结果表明，RepA通过与典型的携带重复序列的θ型质粒类似的机制在肠球菌质粒p703/5的复制中发挥调控作用。
