Figure 4.
G4-DNA Stabilization Causes Genome-wide R-Loop-Dependent Replication Stress
(A) Rtel1F/F;WT RNH1-GFP MEFs were treated with doxycycline. After 24 h, TMPyP4 was added for 24 h. Cells were then collected and lysed, and whole-cell extracts were analyzed by SDS-PAGE and immunoblotted for GFP, RTEL1, and GAPDH.
(B) Rtel1F/F;WT RNH1-GFP MEFs were treated as in (A), and the interaction between PCNA and RNApolII was assessed by PLA. Right: representative images of PLA. Left: quantification of PLA (n = 3).
(C) Rtel1F/F;WT RNH1-GFP MEFs were treated as in (A), and DNA fiber assay was performed. Top: experimental setup. Bottom: distribution of replication fork speeds of DNA fibers. Data are represented as mean ± SE (n = 3).
(D) Left: scatterplot of fork asymmetry of DNA fibers as prepared in (C). Right, bottom: quantification of fork asymmetry of DNA fibers as prepared in (C). In boxplots, horizontal line denotes the mean; whiskers denote the 5th and 95th percentiles (n = 3).
(E) Rtel1F/F;WT RNH1-GFP MEFs were treated with doxycycline. After 24 h, TMPyP4 was added for 24 h and then removed. Cells were incubated for another 24 h and then fixed. The percentage of cells with micronuclei was quantified.
(F) Right: TMPyP4 colony survival assay in Rtel1F/F;WT RNH1-GFP MEFs. Data are mean ± SEM normalized to untreated cells (n = 3). Left: representative images of colonies.
The pvals were determined by unpaired t test, with ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001. Scale bars, 10 μm.