Analysis of initiatin and its regulation in in vitro replication system of plasmid DNA

质粒pSC101基因组编码一个蛋白质（Rep），该蛋白质在其自身DNA的起始中起特殊作用。我们利用基因操作技术开发了该蛋白的超产系统。本研究纯化了Rep蛋白，并对其进行了如下表征。利用多种柱层析技术从过量生产者中获得了高纯度的Reo蛋白。用大肠杆菌粗提物ⅱ段进行体外DNA复制试验。然而，当含有大肠杆菌染色体复制起点的oriC质粒在同一片段中复制时，没有观察到依赖Rep蛋白的pSC101 DNA的复制，这表明该片段的复制机制是活跃的。采用凝胶阻滞法、dna酶i足迹法和核酸外切酶iii酶切法测定了pSC101复制起点（ori）中Rep蛋白的结合位点。在任何实验中，当Rep二聚体/DNA摩尔比为1比3时，在低浓度下，Rep蛋白优先结合与Rep基因启动子区域重叠的反向重复序列。Rep二聚体与dna的摩尔比大于50时，可与起始复制所必需的ori区重复序列结合。Rep蛋白可以在较低浓度下特异性抑制体外转录依赖于Rep启动子。这些结果使我们得出结论，本研究纯化的Rep蛋白保留了作为Rep基因表达抑制因子的功能，而不是作为DNA复制的启动器。要做到这一点，蛋白质可能必须改变其构象或与来自宿主细胞的其他因子形成复合物。

The plasmid pSC101 genome codes a proten (Rep) which functions specifically in initiation of its own DNA. We have developed on overproducing system of the protein using gene manipulation technique. In this study, Rep protein was purified and characterized as follows.1. Reo protein was highly purified with various kinds of column chromatography from the over-producer. In vitro DNA replication was tried with the Fraction II from the E. coli crude extract. However, no replication of pSC101 DNA dependent on Rep protein was observed while oriC plasmid containing the replication origin of the E. coli chromosome was replicated in the same fraction, suggesting that replication machinaries of the fraction were active.2. Binding loci of Rep protein in the replication origin (ori) of pSC101 were determined with gel-retardatin assay, DNase I-footprinting method and exonuclease III-digestion method. In any experiment, it was observed that Rep protein binded preferentially to inverted repeat sequences overlapping to the promoter region of the rep gene at low concentrations as Rep dimer/DNA molar ratios were 1 to 3. Rep protein could bind to the repeated sequences in the ori region, which were indispensable to replication initiation, at more than 50 of molar ratio of Rep dimer to DNA.3. Rep protein could repress specifically in vitro transcription dependent on the rep promoter at the lower concentration.These results lead us a conclution that Rep protein purified in this study retains the function as a repressor for rep gene expression but not as an initiator of DNA replication. To do so, the protein might have to change its conformation or form a complex with other factor(s) from host cells.