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1.
Figure 7

Figure 7. R-loop-promoted mutagenesis requires ssDNA endonuclease TraI.. From: R-loops and nicks initiate DNA breakage and genome instability in non-growing Escherichia coli.

The abundance of R-loops caused by deletion of rnhA is not sufficient, but also requires TraI-generated ssDNA nicks, to promote mutagenesis. Therefore generation of DSEs by R-loops requires both an R-loop and a ssDNA nick. Strains: WT, SMR4562; ΔrnhA, PJH683; ΔtraI, PJH234; ΔrnhA ΔtraI, PJH 963. Mean±s.e.m. of three experiments.

Hallie Wimberly, et al. Nat Commun. 2013 July 5;4:2115.
2.
Figure 3

Figure 3. Loss of RNase HI increases amplification and point mutation.. From: R-loops and nicks initiate DNA breakage and genome instability in non-growing Escherichia coli.

(a,b) Mfd is required for approximately half of amplification and point mutation in rnhA+ (‘wild-type’) cells and all the increase in point mutation and amplification in RNase HI-defective (ΔrnhA) cells. Gene amplification and point mutation in (a) and (b); wild-type (♦), ΔrnhA (), mfd (), and mfd ΔrnhA ( × ) strains SMR4562, PJH683, PJH813 and PJH946. (c) Increased point mutation in cells lacking RNase HI requires DinB/DNA Pol IV, as in RNase HI-proficient cells43. Wild-type (♦), ΔrnhA (), dinB10 (♦), dinB10 ΔrnhA () strains SMR4562, PJH683, SMR5830 and PJH791. Error bars, one s.e.m. of four parallel cultures. These experiments were performed three times with comparable results.

Hallie Wimberly, et al. Nat Commun. 2013 July 5;4:2115.
3.
Figure 6

Figure 6. Gam prevents R-loops from initiating amplification and point mutation.. From: R-loops and nicks initiate DNA breakage and genome instability in non-growing Escherichia coli.

The increased amplification and mutation seen in cells with increased R-loops due to knock out of RNase HI (ΔrnhA) is prevented by doxycycline-induced production of Gam protein (Gam-On) (Methods). Gam binds DSEs45, prevents them engaging in repair46 and reduces (a) point mutation and (b) amplification, confirming that DSEs are required for both pathways. Doxycycline-induced production of Gam protein (Gam-On) prevents the increase in a point mutation and b amplification caused by knock out of RNase HI (ΔrnhA), demonstrating that the increased R-loops cause increased amplification and point mutation via DSEs. Strains: ΔrnhA tetR PJH2039 (); ΔrnhA PN25tetOgam tetR, PJH2443 (); PN25tetOgam tetR, PJH2023 ().Gam-On induction, broken lines. Gam-Off, the same strains as ‘Gam-On’ without doxycycline induction; mock-On, strains with the Tet repressor but without the gam gene treated with doxycycline. Error bars represent one s.e.m. of four parallel cultures. This experiment was performed three times with comparable results.

Hallie Wimberly, et al. Nat Commun. 2013 July 5;4:2115.
4.
Figure 8

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.

Hallie Wimberly, et al. Nat Commun. 2013 July 5;4:2115.
5.
Figure 4

Figure 4. Inhibition of translation increases amplification and point mutation.. From: R-loops and nicks initiate DNA breakage and genome instability in non-growing Escherichia coli.

Gene amplification (a) and point mutation (b) in WT (SMR4562) and mfd (PJH813) strains pulsed with spectinomycin (spec) (broken lines). WT+spec (), WT (♦), mfd+spec (), mfd (). (c) The spectinomycin treatment-induced increase in point mutagenesis is DinB/Pol IV-dependent. Point mutation in WT (SMR4562) and dinB (SMR5830) cells pulsed with spec. WT+spec (), WT (♦), dinB+spec (), dinB (). The curves for the spectinomycin-treated WT cells differ from those of the untreated WT cells significantly, WT and spec-treated WT differ (for three experiments, P=0.002 for point mutation and 0.001 for amplification, Student’s t-test); mfd and spec-treated mfd are not significantly different (P=0.6 for point mutation, and 0.7 for amplification). (d) Spectinomycin treatment does not increase Lac+ revertants in cells overproducing RNase HI. Vector pBAD18 (), vector pBAD18 spec-treated (), pBAD18-rnhA (), pBAD18-rnhA spec-treated (); broken lines denote spectinomycin treatment. Error bars represent one s.e.m. of four parallel cultures. These experiments were performed three times with comparable results.

Hallie Wimberly, et al. Nat Commun. 2013 July 5;4:2115.
6.
Figure 2

Figure 2. Overproduction of RNase HI reduces stress-induced amplification and point mutation.. From: R-loops and nicks initiate DNA breakage and genome instability in non-growing Escherichia coli.

(a) Gene amplification and point mutation in strain PJH1093, carrying prnhA (pBAD18-rnhA). Production of RNase HI is uninduced () (solid line) or induced () (broken line) by the presence of arabinose. Also strain PJH1091, carries empty vector pBAD18 as a control, with transcription from the PBAD promoter induced (♦) or uninduced () by presence or absence of arabinose respectively. (b) Viability and retention of pBAD18-rnhA by day 5 of the experiment shows no plasmid loss. Viability and plasmid loss plots are offset by 2 days: the time needed to form a visible Lac+ colony. Error bars, one s.e.m. of four parallel cultures. These and all experiments were performed three times with comparable results.

Hallie Wimberly, et al. Nat Commun. 2013 July 5;4:2115.
7.
Figure 5

Figure 5. R-loops are not needed for mutagenesis when DSBs are caused by I-SceI.. From: R-loops and nicks initiate DNA breakage and genome instability in non-growing Escherichia coli.

Overproduction of RNase HI (reduction in R-loops) does not decrease mutagenesis when I-SceI-generated DSBs are provided. (a) The RNase HI-overproducing strain (PJH1239) (♦) shows about half as much Lac+ mutation as the strain carrying the control plasmid (PJH1237) (). (b) The DSB-producing strain (carrying the chromosomal inducible I-SceI enzyme and I-SceI cutsite near lac) that over-produces RNase HI (PJH1321) (♦) shows no reduction of Lac+ mutants compared with the isogenic strain carrying the control plasmid (PJH1319) (), indicating that R-loops are not necessary for mutation when DSBs are provided. This result holds when both I-SceI enzyme and cutsite are present and not in strains with the enzyme only (a). (c) Strains experiencing double-strand cutting by I-SceI show reduced viability. However, the viability decrease is the same for cells with the rnhA plasmid as for those with the control plasmid making quantitative comparisons meaningful. Relative viability was determined per21. Values in (a,b) have not been corrected for declining viability. Error bars represent one s.e.m. of four parallel cultures. Each experiment was performed three times with comparable results.

Hallie Wimberly, et al. Nat Commun. 2013 July 5;4:2115.
8.
Figure 1

Figure 1. Model for transcription-promoted R-loop initiation of DSEs.. From: R-loops and nicks initiate DNA breakage and genome instability in non-growing Escherichia coli.

(ac) R-loops form by incorporation of the transcript (red) into supercoiled DNA (blue/black) behind the site of transcription41. R-loop formation is usually inhibited by ribosomes or the R-loop removed by RNase HI. (b) Stalled RNAP (circle) (c) is removed by Mfd38. (d) The R-loop can then form a replication fork35. (e) If the replication fork encounters a nick in a template DNA strand, the fork will collapse (f), forming a single DSE12. In stressed cells, the collapsed fork might be repaired and restarted by microhomology-mediated break-induced replication (gj)26 producing genome rearrangements (duplication shown here). (g) DSE degradation and 5′-end resection by RecBCD might be followed by h annealing of the overhanging 3′-end to ssDNA at a site of microhomology (vertical lines), shown here in the lagging-strand template of another replication fork (blue). (i,j) This replication restart is shown at a position behind where the initial fork collapsed, so that a segment of the genome including the lac region becomes duplicated. The duplication can be expanded into an amplified array by unequal crossing-over (not illustrated). (k) Alternatively, point mutation is proposed to occur when the DSE is repaired by homologous recombination-mediated replication-fork restart using error-prone polymerase Pol IV during restart due to licensing of Pol IV (and also Pols II and V) by the RpoS and SOS stress responses92124.

Hallie Wimberly, et al. Nat Commun. 2013 July 5;4:2115.

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