Nature of R-factor replication in the presence of chloramphenicol

Covalently closed circular deoxyribonucleic acid molecules of RSF1030, a nonconjugative R-factor, initiate and complete rounds of semiconservative replication in the absence of protein synthesis long after the bacterial chromosome has ceased its replication. RSF1030 replication under these conditions is sensitive to the inhibitor of ribonucleic acid synthesis, rifampicin. The product of this replication, a covalently closed DNA molecule, shows, in contrast to those molecules produced during the replication in a logarithmically growing culture of Escherichia coli, a transition to the open circular form upon treatment with ribonucleases of alkali. Analysis of the product resulting from alkali treatment indicates that there is a single break in one strand of the original circular duplex. This alkali-sensitive site occurs with equal probability in either of the complementary strands. These results are interpreted as a requirement for an RNA primer for the initiation of RSF1030 DNA synthesis and as showing that its removal from the covalently closed molecule is inhibited in the absence of protein synthesis.