title: R-loops and nicks initiate DNA breakage and genome instability in non-growing Escherichia coli.
abstract: 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.
authors.affiliation: Department of Molecular and Human Genetics, 1 Baylor Plaza, Houston, Texas 77030, USA.
authors.forename: Hallie
authors.initials: H
authors.lastname: Wimberly
authors.forename: Chandan
authors.initials: C
authors.lastname: Shee
authors.forename: P C
authors.initials: PC
authors.lastname: Thornton
authors.forename: Priya
authors.initials: P
authors.lastname: Sivaramakrishnan
authors.forename: Susan M
authors.initials: SM
authors.lastname: Rosenberg
authors.forename: P J
authors.initials: PJ
authors.lastname: Hastings
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development*; D042822:Genomic Instability / Q000235:genetics*; D008957:Models, Genetic; D016296:Mutagenesis / Q000235:genetics; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism*; D017354:Point Mutation / Q000235:genetics; D014176:Protein Biosynthesis; D012260:Ribonucleases / Q000378:metabolism; D013312:Stress, Physiological / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009692:Nucleic Acid Heteroduplexes; D014157:Transcription Factors; D014764:Viral Proteins; C065410:gam protein, Coliphage; C072103:transcription repair coupling factor protein, Bacteria; D012260:Ribonucleases