Isolation and characterization of host mutants defective in msDNA synthesis: role of ribonuclease H in msDNA synthesis

Abstract

In some Escherichia coli strains, a single-stranded DNA (msDNA) covalently linked to RNA is produced by a reverse transcriptase (RT) encoded by a genetic element called a retron. We have looked for host genes involved in msDNA synthesis. From screening of 10,000 mutants generated by Tn5 insertions, we obtained 3 chromosomal mutants defective msDNA synthesis. Analysis showed that all 3 mutants were affected in the gene for ribonuclease H (rnh). In rnh mutants, the reverse transcription products were heterogeneous with several sizes, smaller and bigger than the wild-type msDNA, showing that the reverse transcription is inhibited by the lack of RNase H activity. It also suggests that the RT recognizes the structure of msDNA for the termination of reverse transcription.