Isolation of Supercoiled Colicinogenic Factor E1 DNA Sensitive to Ribonuclease and Alkali

AUTOR(ES)

Blair, D. G.

RESUMO

The synthesis of the covalently-closed, circular DNA form of colicinogenic factor E1 (ColE1) continues in Escherichia coli cells after the addition of chloramphenicol. A large portion of the purified supercoiled ColE1 DNA molecules made in the presence of chloramphenicol are converted to the open circular DNA form after treatment with alkali (pH 13), RNase A, or RNase H. These treatments do not significantly affect the covalently-closed form of ColE1 DNA isolated from normally growing E. coli cells. The open circular product resulting from treatment of supercoiled ColE1 DNA with RNase A possesses a single break in one strand of the circular duplex. The site sensitive to RNase A occurs with equal probability in either of the complementary strands. Both synthesis of ColE1 DNA and the formation of supercoiled ColE1 DNA sensitive to RNase A or alkali are prevented by the inhibitor of RNA synthesis, rifampicin. These results indicate that covalently-closed ColE1 DNA containing one or more ribonucleotides accumulates during ColE1 replication in the presence of chloramphenicol. It is proposed that this incorporated RNA served as a primer during the initiation of synthesis of ColE1 DNA and that its removal from the circular DNA is inhibited in cells incubated in the presence of chloramphenicol.

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