Increased replication fork progression by ISG15 depends on RECQ1 and leads to unrestrained DNA replication and DNA breakages. (A) FLAG-immunoblot on protein extracts derived from cytosolic or nuclear fractions of U2OS FIT cells expressing EV or FLAG-ISG15ΔGG. Lamin A and GAPDH are used as controls of fractionation and loading. (B) FLAG-ISG15 was immunoprecipitated (IP FLAG) from nuclear extracts as in A and analyzed by FLAG immunoblot. Sn, supernatant after immunoprecipitation. (C) FLAG immunoprecipitation as in B, detected by Coomassie blue staining. The band corresponding to FLAG-ISG15ΔGG is indicated. HL and LC represent heavy and light chain of the FLAG antibody, respectively. (D) Anti-HA immunoprecipitation from HEK293T cells expressing HA-RecQ1 and myc-ISG15 followed by immunoblot with the indicated antibodies. TCL, total cell extracts. (E) HEK293T cells were transfected with 2 µg expression constructs of indicated proteins. Immunoblot was used to detect HaloTag, NanoLuc, and GAPDH. (F) RECQ1 protein levels in U2OS FIT expressing FLAG-ISG15 after induction with 1 µg/ml doxycycline (dox) for different time points indicated. Vinculin immunoblot is used as loading control. (G) Effect of 1 µM cisplatin (CDDP) or 0.5 mM HU treatment on U2OS FIT cells expressing EV, FLAG-ISG15, or FLAG-ISG15ΔGG after induction with 1 µg/ml doxycycline for 48 h. UN, untreated. Drug effect (IdU + drug) is normalized on CldU track length in untreated conditions. At least 100 tracks were scored per sample (n = 3). (H) Neutral comet assay to measure DNA double-strand breaks in U2OS FIT cells expressing EV, FLAG-ISG15, or FLAG-ISG15ΔGG after induction with 1 µg/ml doxycycline for 7 d and treated with 50 nM CPT (n = 3). Statistical analysis according to Mann–Whitney test; ****, P < 0.0001.