Characterization of the ColE2-like replicon of plasmid pTT8 from Thermus thermophilus.
We identified the 1.6-kb region of Thermus thermophilus plasmid pTT8 capable of autonomous replication, which shows a significant sequence similarity to the replicon regions of the ColE2-related plasmids. We showed the requirement of DNA polymerase I for pTT8 replication. The putative rep gene coding for the replication initiator protein, Rep, similar to those of the ColE2-related plasmids was cloned into an expression vector. The 6xHis-Rep protein expressed in Escherichia coli was successfully purified by stepwise denaturing with urea and refolding in the presence of glycerol on Ni-resin. We identified the nucleotide sequence recognized by the pTT8 Rep protein by the SELEX experiment using the purified protein, and proposed the existence of the third origin of pTT8 replication different from those predicted previously.
Aoki
Kazuteru
K
Department of Biology, Faculty of Science, Shinshu University, Matsumoto, Nagano 390-8621, Japan.
Itoh
Tateo
T
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D004265:DNA Helicases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004261:DNA Replication; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000235:genetics*; D018741:Replication Origin; D012093:Replicon / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D016963:Thermus thermophilus / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D004259:DNA-Directed DNA Polymerase; D004265:DNA Helicases