Synthesis and repair of RNA-containing supercoiled plasmid ColE1 DNA in Escherichia coli

Supercoiled plasmid molecules sensitive to nicking by RNase or alkali have been shown to accumulate during replication of colicinogenic factor E1 (ColE1) in Escherichia coli in the presence of chloramphenicol. The possibility that this sensitivity is due to the covalent integration of RNA molecules during the synthesis of plasmid DNA is supported by the demonstration that (a) strands of supercoiled ColE1 newly replicated in the presence of chloramphenicol exhibit sensitivity to RNase and alkali treatment, while (b) RNase- and alkali-resistant circular strands of plasmid DNA synthesized either before or after the addition of chloramphenicol remain resistant during subsequent replication of the plasmid in the presence of chloramphenicol. Furthermore, newly made plasmid DNA strands cannot act as templates for further rounds of replication if they possess an RNA segment. The existence of a repair mechanism for the removal of the RNA segment from supercoiled ColE1 DNA molecules was demonstrated by pulse-chase experiments. It was observed that the proportion of RNase-sensitive molecules is considerably higher in pulse-labeled as compared to continuously labeled ColE1 DNA synthesized in the presence of chloramphenicol, and the proportion of pulse-labeled ColE1 DNA that is RNase sensitive is greatly reduced during a chase period. Removal of the RNA segment is also carried out effectively at the restrictive temperature in temperature-sensitive DNA polymerase I mutants. In a survey of other bacterial mutants defective in the repair of damaged DNA, a substantial increase in the rate of accumulation of RNase-and alkali-sensitive supercoiled ColE1 DNA in the presence of chloramphenicol was observed in recBC and uvrA mutants in comparison with the wild-type strains.