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

PSC101基因组编码一个蛋白(Rep)，该蛋白在启动自身DNA的过程中起着特殊的作用。我们利用基因操作技术建立了蛋白质的高产体系。本研究对Rep蛋白进行了纯化，并对其进行了鉴定。通过各种柱层析，从高产菌株中获得了高纯度的Reo蛋白。用从大肠杆菌粗提物中提取的组分II在体外进行DNA复制。然而，当含有大肠杆菌染色体复制起点的ORIC质粒在同一组分中复制时，没有观察到依赖于Rep蛋白的pSC101 DNA的复制，这表明该组分的复制机制是活跃的。用凝胶延迟法、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)上Rep蛋白的结合位点。在实验中观察到，当Rep二聚体/DNA摩尔比为1：3时，Rep蛋白优先与Rep基因启动子区域重叠的反向重复序列结合。当Rep二聚体与DNA摩尔比大于50时，Rep蛋白可与复制起始所必需的OrI区重复序列结合。结果表明，本研究纯化的Rep蛋白具有抑制rep基因表达的功能，但不能作为DNA复制的启动子。要做到这一点，蛋白质可能不得不改变其构象，或者与宿主细胞中的其他因子(S)形成复合体。

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.