PHF6 prevents replication-associated damage and accumulation of genomic rearrangements at the FRA16D chromosome fragile site. (A) Locus map of CFS-FRA16D SbfI digested segment. The fluorescence in situ hybridization probes that identify the segment are labeled in blue. (B) Aligned photomicrograph images of labeled DNA molecules from Jurkat PHF6 infected with an empty vector control (EV, control sgRNA) or with an sgRNA targeting the second PHD2 domain of PHF6 (PHF6 PHF6 KO). The yellow arrows indicate the sites along the molecules where the iododeoxyuridine transitioned to chlorodeoxyuridine. White rectangles indicate representative sites of replication fork pausing. The molecules are arranged in the following order: molecules with initiation events, molecules with 3′ to 5′ progressing forks, molecules with 5′ to 3′ progressing forks, and molecules with termination events. The quantification in the upper right panel shows the percentage of molecules with rearrangements at CFS-FRA16D in Jurkat PHF6 EV (blue bar) and Jurkat KO (red bar). Error bars represent mean ± standard deviation from data collected from 2 independent experiments. The quantification in the lower right panel shows the replication fork speed at CFS-FRA16D in Jurkat PHF6 EV (blue bar) and Jurkat KO (red bar). Error bars represent mean ± standard deviation from data collected from 2 independent experiments. (C) Close-up of the 5′ to 3′ region of CFS-FRA16D showing aberrant probe patterns in individual DNA molecules.