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

点击翻译按钮获取中文摘要

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.