Nucleotide sequences required for a ColE1-type plasmid to replicate in Escherichia coli cells with or without RNase H.
To elucidate the replication mechanism of a ColE1-type plasmid in RNase H-deficient (rnh-) strains of Escherichia coli, we constructed plasmid derivatives that deleted the whole, or a part, of the 5'-AAAAA-3' sequence (positions -3 to +2) that acts as the origin of replication in vivo and in vitro in the presence of RNase H. The activity of plasmid replication in rnh+ cells was found to be reduced by alterations of the AAAAA sequence. The activity could be restored when the derivatives, retaining the upstream sequence down to -8, regained a sequence containing at least two A residues in the region from -3 to +2. By contrast, replication in rnh- cells was maintained at high levels even when the deletion included the AAAAA sequence and extended up to position -7. The activity in rnh- cells decreased as deletions proceeded to -8 and further up to -17, and was abolished completely by further upward deletions. We concluded that in rnh- cells the plasmid replicates by a mechanism that operates only when RNase H is inactive. This RNase H-sensitive replication in rnh- cells seems to require the RNA-DNA hybrid formation that is also required for RNase H-dependent replication in rnh+ cells. The hybrid formation probably contributes by unwinding a portion of DNA from which replication can be initiated.
Ohmori
H
H
Institute for Virus Research, Kyoto University, Japan.
Murakami
Y
Y
Nagata
T
T
D001483:Base Sequence; D003087:Colicins; D004261:DNA Replication; D004269:DNA, Bacterial; D004722:Endoribonucleases / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D016914:Ribonuclease H
D003087:Colicins; D004269:DNA, Bacterial; D004722:Endoribonucleases; D016914:Ribonuclease H