The Mechanism of Initiation of DNA Replication of Drug Resistance Plasmid R100

抗性质粒R100的DNA复制的起始基本上需要两个因素，起始蛋白RepAl和DNA复制起点ori。已知R100的复制从ori单向进行。本研究在此期间的主要研究成果如下：1.我们试图阐明R100单向复制的分子机制。利用体外DNA复制系统，我们发现DNA复制的方向完全依赖于ori区的方向。然后，我们鉴定了复制中间质粒分子中存在的前导链和滞后链DNA，并确定了前导链DNA的5 ′-末端和滞后链DNA的3 ′-末端。根据所得结果，我们提出了R100单向DNA复制的以下模型：（1）在复制起始的必需区域内合成一个引物RNA，以启动引导链DNA的合成。(2)随后，滞后链DNA被合成，但在ori区域内的独特位点处终止，导致滞后链DNA合成的强烈停止。(3)前导链的合成继续单向进行，可能与其他滞后链DNA的合成合作。我们化学合成了149 bp的ori序列，它是ori的一部分，不含danA盒序列。我们发现该序列不能作为ori发挥作用，这表明dnaA盒对R100复制的启动是重要的。我们还发现，在这个区域的DNA是1.3。在ori下游约2kb的区域，我们发现了负责R100复制稳定维持的新基因（命名为pemA和pemB）。pemA和pemB的基因产物分别被鉴定为9 K和12 K蛋白。

Initiation of DNA replication of resistance plasmid R100 requires basically two factors, an initiator protein RepAl and an origin of DNA replication, ori. Replication of R100 is known to proceed unidirectionally from ori. Our studies during this grant period can be summarized as follows:1. We tried to elucidate molecular mechanisms of unidirectional replication of R100. Using in vitro DNA replication system, we showed that the direction of DNA replication is solely dependent on the orientation of the ori region. Then, we identified the leading strand and lagging strand DNA present in the replication intermediate plasmid molecules and determined the 5'-end of the leading strand DNA and 3'-end of the lagging strand DNA. Based on the results obtained, we proposed the following model for the unidirectional DNA replication of R100; (1) A primer RNA is synthesized within the essential region for initiation of replication to initiate synthesis of the leading strand DNA. (2) Subsequently, the lagging strand DNA is synthesized, but is terminated at unique sites within the ori region, causing a strong stop for lagging strand DNA synthesis. (3) Leading strand synthesis continues unidirectionally, possibly cooperated with synthesis of the other lagging strand DNA.2. We chemically synthesized the 149 bp of ori sequence which is apart of ori and does not contain a danA box sequence. We found that the sequence was not able to function as ori, suggesting that the dnaA box is important for initiation of R100 replication. We also found that DNA in this region was bent.3. In the region about 2 kb downstream from ori, we found the novel genes (named pemA and pemB) responsible for the stable maintenance of R100 replication. The gene products of pemA and pemB were identified as 9 K and 12 K proteins, respectively.