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

• DOI: 10.1038/ncomms3115
• Publication Date: 2013-07-05T10:13:19Z
• Authors: Hallie Wimberly; Chandan Shee; P. C. Thornton; Priya Sivaramakri...; Susan M. Rosenberg; P. J. Hastings
• Keywords: 0301 basic medicine; Models, Genetic; Transcription, Genetic; Nucleic Acid Heteroduplexes; DNA, Single-Stranded; Article; Genomic Instability; 3. Good health; DNA-Binding Proteins; Viral Proteins; 03 medical and health sciences; Ribonucleases; Bacterial Proteins; Mutagenesis; Stress, Physiological; Protein Biosynthesis; Escherichia coli; Point Mutation; DNA Breaks, Double-Stranded; DNA Damage; Transcription Factors

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.