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.
