Figure 8: Formation of DSEs promoted by R-loops also requires ssDNA nicks.
From: R-loops and nicks initiate DNA breakage and genome instability in non-growing Escherichia coli
(a) Quantification of cells with GamGFP foci. Cells carrying the F′ plasmid show more foci than F− (WT) cells, and these extra foci are TraI-dependent, and thus result from the ssDNA nicks made by TraI endonuclease at oriT. Cells lacking RNase HI show more foci than their isogenic RNase HI+ parents (compare WT with ΔrnhA; and [F′] with ΔrnhA[F′]) indicating that lack of RNase HI (increased R-loops) causes increased DSEs. Finally, the increased foci caused by loss of RNase HI in the F′ (compared with that in F− ΔrnhA cells) requires TraI ssDNA endonuclease. Therefore, R-loop-mediated foci require both the R-loop and a ssDNA nick. Means±s.e.m. of three experiments. WT ‘wild-type’ SMR14015; [F′] SMR16387; [F′ΔtraI] SMR16475; ΔrnhA SMR16379; ΔrnhA [F′] SMR16389; ΔrnhA [F′ΔtraI] SMR16477. All strains carry a chromosomal tetR and PN25tetOgam-gfp. Supplementary Table S2 for numerical values. (b) Examples of fields of cells showing few GamGFP foci in wild-type, SMR14015, and multiple foci in most cells in ΔrnhA [F′], SMR16389.
