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.

Role for RNA:DNA hybrids in origin-independent replication priming in a eukaryotic system

“…S4B). This result supports the idea that the concomitant loss of RNase H and Top1 activities has an additive effect on RNA:DNA hybrid formation (13,33).…”

“…Additional evidence for R-loop-primed replication was given by the observation that rnhA mutants are prone to an increase in mutation and DNA amplification events if origin activity is suppressed. These events required removal of the RNA polymerase (RNAP) to allow conversion of an R-loop into a replication fork (33). In summary, R-loops may act as the earliest known mutagenic intermediate in transcribed regions, and accelerate adaptation to genomic stress in prokaryotes.…”

Roles of Nucleoid-Associated Proteins in Stress-Induced Mutagenic Break Repair in StarvingEscherichia coli

“…Our discovery here of four NAPs that promote MBR (Fis, H-NS, CspE, and CbpA) (Figure 1), in addition to the four NAPs identified in the previous screen [Lrp, Hfq, CspC, and IHF (Al Mamun et al 2012)], implies that the full MBR network is likely to be about twice as large as the 93 genes of Al Mamun et al (2012)-180-190 genes. In addition to the four NAPs added in this work, we also subsequently identified the transcription protein Mfd (Wimberly et al 2013) as MBR promoting. Also Figure 6 The updated mutagenic break-repair network with NAPs.…”

“…E. coli cells carrying a leaky lacIZ +1-bp frameshift allele can revert either by a single-base deletion compensatory frameshift mutation (SNV) or by amplification of the leaky lac allele to give a tandem array of 20 or more copies (GCR) (Hastings et al 2000Hersh et al 2004;Rogers et al 2015). TraI single-strand endonuclease makes nicks in the F9 that become DSBs (Ponder et al 2005;Wimberly et al 2013), which are required for both SNV and GCR (Harris et al 1994;Wimberly et al 2013).…”

“…Downstream of SOS, solid red; DNA polymerases, green circle; ROS level maintenance, solid yellow; replicon copy number, solid salmon; NAPs, blue circle; unknown function in MBR, solid gray (Gibson et al 2015). This rendition includes the NAPs shown here to promote MBR, and the other proteins discovered to promote MBR after the initial description of the MBR network by Al Mamun et al (2012): Mfd (Wimberly et al 2013) and PhoB and PhoR (Gibson et al 2015). discovered after the 93-gene network of Al Mamun et al (2012), PhoB and PhoR promote MBR by an as yet unknown mechanism (Gibson et al 2015).…”