Determination of the nick site at oriT of IncI1 plasmid R64: global similarity of oriT structures of IncI1 and IncP plasmids.
The nick site at the origin of transfer, oriT, of IncI1 plasmid R64 was determined. A site-specific and strand-specific cleavage of the phosphodiester bond was introduced during relaxation of the oriT plasmid DNA. Cleavage occurred between 2'-deoxyguanosine and thymidine residues, within the 44-bp oriT core sequence. The nick site was located 8 bp from the 17-bp repeat. A protein appeared to be associated with the cleaved DNA strand at the oriT site following relaxation. This protein was observed to bind to the 5' end of the cleaved strand, since the 5'-phosphate of the cleaved strand was resistant to the phosphate exchange reaction by polynucleotide kinase. In contrast, the 3' end of the cleaved strand appeared free, since it was susceptible to primer extension by DNA polymerase I. The global similarity of the oriT structures of IncI1 and IncP plasmids is discussed.
Department of Biology, Tokyo Metropolitan University, Japan.
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Furuya
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Komano
D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004270:DNA, Circular / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000502:physiology; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D014161:Transduction, Genetic / Q000235:genetics*
D004269:DNA, Bacterial; D004270:DNA, Circular; D004268:DNA-Binding Proteins; D012967:Sodium Dodecyl Sulfate; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase; D010450:Endopeptidases