R-loops and nicks initiate DNA breakage and genome instability in non-growing Escherichia coli

Nat Commun. 2013:4:2115. doi: 10.1038/ncomms3115.


Double-stranded DNA ends, often from replication, drive genomic instability, yet their origin in non-replicating cells is unknown. Here we show that transcriptional RNA/DNA hybrids (R-loops) generate DNA ends that underlie stress-induced mutation and amplification. Depleting RNA/DNA hybrids with overproduced RNase HI reduces both genomic changes, indicating RNA/DNA hybrids as intermediates in both. An Mfd requirement and inhibition by translation implicate transcriptional R-loops. R-loops promote instability by generating DNA ends, shown by their dispensability when ends are provided by I-SceI endonuclease. Both R-loops and single-stranded endonuclease TraI are required for end formation, visualized as foci of a fluorescent end-binding protein. The data suggest that R-loops prime replication forks that collapse at single-stranded nicks, producing ends that instigate genomic instability. The results illuminate how DNA ends form in non-replicating cells, identify R-loops as the earliest known mutation/amplification intermediate, and suggest that genomic instability during stress could be targeted to transcribed regions, accelerating adaptation.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Bacterial Proteins / metabolism
  • DNA Breaks, Double-Stranded
  • DNA Damage*
  • DNA, Single-Stranded / metabolism
  • DNA-Binding Proteins / metabolism
  • Escherichia coli / genetics*
  • Escherichia coli / growth & development*
  • Genomic Instability / genetics*
  • Models, Genetic
  • Mutagenesis / genetics
  • Nucleic Acid Heteroduplexes / metabolism*
  • Point Mutation / genetics
  • Protein Biosynthesis
  • Ribonucleases / metabolism
  • Stress, Physiological / genetics
  • Transcription Factors / metabolism
  • Transcription, Genetic
  • Viral Proteins / metabolism


  • Bacterial Proteins
  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • Nucleic Acid Heteroduplexes
  • Transcription Factors
  • Viral Proteins
  • gam protein, Coliphage
  • transcription repair coupling factor protein, Bacteria
  • Ribonucleases