A subset of G4 motifs have high Cdc20 binding indicative of replication fork pausing in Pfh1-depleted cells. (A) The Venn diagram gives the overlaps between G4 motifs associated with high Pfh1 (orange circle), high Cdc20 in WT cells (green circle) and high Cdc20 in Pfh1-depleted cells (blue circle). For the most part, G4 motifs with high Cdc20 in WT are a subset of those with high Cdc20 in Pfh1-depleted cells. (B) To compare the overall Cdc20 occupancy at G4 motifs in Pfh1-expressing (blue) and Pfh1-depleted cells (red), we estimated the expected distribution of high Cdc20 sites for 1,000 control region sets matched to the observed G4 motifs (histograms). Because the coverage of the genome by Cdc20 is higher in Pfh1-depleted cells, the expected G4 motif association is somewhat higher for Pfh1-depleted cells (58.3; red) than Pfh1-expressing cells (50.0; blue). In Pfh1-expressing cells the 50 G4 motifs with high Cdc20 occupancy (blue arrow) was not different from the expected number (P = 0.529). In Pfh1-depleted cells, significantly more G4 motifs were associated with high Cdc20 regions than expected (red arrow; 77 regions; P = 0.003). (C) Cdc20-3HA peaks were validated in WT and Pfh1-depleted cells by ChIP-qPCR using an anti-HA antibody as described for Figure 1C. In Pfh1-depleted cells, all G4 motifs and the tRNA gene had a significant increase of Cdc20 levels compared to WT cells (two-tailed student t-test, P ≤ 0.04). ChIP, chromatin immunoprecipitation; G4, G-quadruplex; qPCR, quantitative PCR; WT, wild type.