Fig. 3: USP37 depletion induces premature CMG unloading by TRAIP during topological stress.

a Plasmid DNA was incubated in the indicated egg extracts in the presence or absence of ICRF-193. At specified times, chromatin was recovered and immunoblotted for the indicated proteins. USP37 depletion efficiency is shown in Supplementary Fig. 6a. Ub-MCM7, ubiquitylated MCM7; Ub-MCM6, ubiquitylated MCM6; Ub-CDC45, ubiquitylated CDC45. See also Supplementary Fig. 6b. b Plasmid DNA was incubated in the indicated egg extracts in the presence of ICRF-193 and, where indicated, p97-i. Extracts were supplemented with recombinant WT or catalytically inactive (C347S) USP37 expressed in wheat germ extract (WGE), or WGE with empty vector (EV). At specified times, chromatin was recovered and immunoblotted for the indicated proteins. Samples are from the same experiment; blots were processed in parallel. ΔM, ΔMOCK; Ub-MCM4, ubiquitylated MCM4. See also Supplementary Fig. 6c, d. c As in (b), but where indicated, TRAIP was co-depleted with USP37. Egg extracts were supplemented with recombinant WT TRAIP expressed in WGE, or WGE with EV. Samples are from the same experiment; blots were processed in parallel. Depletion efficiencies and levels of recombinant proteins are shown in Supplementary Fig. 6e. See also Supplementary Fig. 6f. d Representative pictures of CDC45-EdU PLA. Scale bar: 10 μm (top); Dot plot indicating the number of PLA foci between CDC45 and EdU (replicating DNA) in untreated or camptothecin-treated cells (bottom). Untreated: n = 441 (CTRL, sgCTRL), 389 (CTRL, sgTRAIP), 457 (USP37 KO10, sgCTRL), 363 (USP37 KO10, sgTRAIP), 386 (USP37 KO19, sgCTRL), 385 (USP37 KO19, sgTRAIP) cells; camptothecin: n = 450 (CTRL, sgCTRL), 361 (CTRL, sgTRAIP), 449 (USP37 KO10, sgCTRL), 409 (USP37 KO10, sgTRAIP), 369 (USP37 KO19, sgCTRL), 372 (USP37 KO19, sgTRAIP) cells. Cells were analyzed from three biological replicates. Bar represents the median of three independent experiments. Dashed cyan and brown lines are the position of the median for untreated and camptothecin-treated CTRL, sgCTRL cells, respectively. Black dots indicate means in each independent experiment. Statistics indicate two-tailed Wilcoxon rank-sum tests for p values without adjustment for multiple hypothesis testing and Cohen’s d for effect sizes (γ) (see methods). Source data are provided as a Source Data file.