G4 motifs are significantly associated with DNA damage. (A) The Venn diagram shows the overlaps between G4 motifs that were associated with high Pfh1 (orange), high γ-H2A in WT cells (green circle), and high γ-H2A in Pfh1-depleted cells (blue circle). Nearly all G4 motifs with high γ-H2A in WT also had high γ-H2A in Pfh1-depleted cells. (B) G4 motifs were significantly associated with high γ-H2A in both Pfh1-expressing (blue) and Pfh1-depleted (red) cells. The significance of these associations was determined by comparison to the number of overlaps in 1,000 random control sets in both Pfh1-expressing (blue histogram) and Pfh1-depleted cells (red histogram). When Pfh1 was depleted, there was a dramatic increase in the amount of DNA damage at G4 motifs, as inferred from γ-H2A associations for Pfh1-depleted (155.6; red) versus Pfh1-expressing (61.8; blue) cells. In both contexts, the association of G4 motifs with high γ-H2A was significant (blue arrow; Pfh1-expressing, P = 0.021; red arrow; Pfh1-depleted, P = 0.014). (C) γ-H2A associated peaks were validated in WT and Pfh1-depleted cells by ChIP-qPCR using an anti- γ-H2A antibody as described in Figure 1C except that the primer pairs were located further from each test site (within 5 kb) to detect regions with the highest γ-H2A content. By two-tailed student t-test, all G4 motifs and the tRNA gene had significantly increased γ-H2A in Pfh1-depleted cells compared to WT cells (P ≤ 0.05). ChIP, chromatin immunoprecipitation; G4, G-quadruplex; qPCR, quantitative PCR; WT, wild type.