Figure 3From: The essential Schizosaccharomyces pombe Pfh1 DNA helicase promotes fork movement past G-quadruplex motifs to prevent DNA damageG4 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.Back to article page