USP37 prevents premature disassembly of stressed replisomes by TRAIP

Abstract

The eukaryotic replisome, which consists of the CDC45-MCM2-7-GINS (CMG) helicase, replicative polymerases, and several accessory factors, sometimes encounters proteinaceous obstacles that threaten genome integrity. These obstacles are targeted for removal or proteolysis by the E3 ubiquitin ligase TRAIP, which associates with the replisome. However, TRAIP must be carefully regulated to avoid inappropriate ubiquitylation and disassembly of the replisome. Here, we demonstrate that human cells lacking the de-ubiquitylating enzyme USP37 are hypersensitive to topoisomerase poisons and other replication stress-inducing agents. Furthermore, TRAIP loss rescues the hypersensitivity of USP37 knockout cells to topoisomerase inhibitors. In Xenopus egg extracts depleted of USP37, TRAIP promotes premature CMG ubiquitylation and disassembly when converging replisomes stall. Finally, guided by AlphaFold-Multimer, we discovered that binding to CDC45 mediates USP37's response to topological stress. We propose that USP37 protects genome stability by preventing TRAIP-dependent CMG unloading when replication stress impedes timely termination.

Fig. 1. USP37 loss protects cells from topoisomerase poisons- and replication stress-associated DNA damage.

a Rank-plot showing gene enrichment scores (normZ) of CRISPR screen hits upon camptothecin treatment in human U2OS cells. Negative scores represent dropouts, genes whose loss is predicted to promote drug sensitivity. Blue dots indicate well-characterized factors affecting cellular sensitivity to camptothecin; the red dot indicates USP37. b–e Clonogenic survival assays of control (CTRL) and USP37 knockout (KO; results from two independent clones plotted) RPE-1 TP53 KO cells upon treatment with (b) camptothecin, (c) etoposide, (d) ICRF-193, or (e) aphidicolin. n = 4 independent experiments (b–d), n = 3 independent experiments (e). Statistical analysis for (b–e) was performed using two-way ANOVA and Tukey test for multiple comparisons. f Representative images of RPA (green) and γH2AX (orange) staining in S-phase (magenta) positive cells. Scale bar: 20 μm. g, h Quantification of RPA (g) or γH2AX (h) foci in S-phase cells (EdU positive). Cells were treated with the indicated doses of the drugs for 4 h. For (g), untreated: n = 2427 (WT), 1599 (USP37 KO6), 1212 (USP37 KO17) cells; camptothecin: n = 2316 (WT), 1665 (USP37 KO6), 1350 (USP37 KO17) cells; aphidicolin: n = 2538 (WT), 1234 (USP37 KO6), 1164 (USP37 KO17) cells. For (h), same as for (g), but n = 2426 (WT, untreated), 1598 (USP37 KO6, untreated) cells. Cells were analyzed from five biological replicates, except for USP37 KO6 (four biological replicates). Bars represent the median. 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). CPT camptothecin. i–k Clonogenic survival assays of control (CTRL) or USP37 KO (clone 10) cells expressing mCherry (EV), mCherry-USP37^WT or mCherry-USP37^C350A (catalytic inactive) upon treatment with camptothecin (i), etoposide (j), or aphidicolin (k); n = 4 independent experiments (i); n = 5 independent experiments (j), with the exception of “C350A” condition, where n = 4); n = 3 independent experiments (k). Bars represent means ± SEM. Statistical analysis for (i–k) was performed using two-way ANOVA and Šídák test for multiple comparisons. Source data are provided as a Source Data file.

Fig. 2. Genetic screen unveils functional connections between USP37 and TRAIP.

a Biplot showing gene enrichment scores (normZ) reflecting viability in untreated conditions of WT (y-axis) and USP37 KO (x-axis) U2OS cells. b U2OS USP37 WT cells (left histogram) and USP37 KO cells (right histogram) were transfected with a CRISPR sgRNA targeting TRAIP and samples were collected 4, 7, 11, and 15 days afterwards and subjected to TRAIP sequence analysis. Percentages of unedited or edited cells at the TRAIP locus at the indicated time points are plotted. n = 3 independent experiments. Bars represent means ± SEM. c–e Clonogenic survival assays of CTRL or USP37 KO RPE-1 TP53 KO cells transduced with a LacZ control sgRNA or with a sgRNA targeting TRAIP upon treatment with camptothecin (c), ICRF-193 (d), and aphidicolin (e). n = 3 independent experiments. Bars represent means ± SEM. Half plot points indicate zero percent viability. Statistical analysis for (c–e) was performed using two-way ANOVA and Tukey test for multiple comparisons. f Representative images of RPA (green) and γH2AX (orange) staining in S-phase (magenta) positive cells. Scale bar: 20 μm. g, h Quantification of RPA (g) or γH2AX (h) foci in S-phase cells (EdU positive). Cells were treated with 20 nM camptothecin for 4 h. For (g, h), untreated: n = 1747 (CTRL, siCTRL), 1262 (CTRL, siTRAIP), 865 (USP37 KO6, siCTRL), 533 (USP37 KO6, siTRAIP), 753 (USP37 KO17, siCTRL), 458 (USP37 KO17, siTRAIP); camptothecin: n = 1885 (CTRL, siCTRL), 1315 (CTRL, siTRAIP), 838 (USP37 KO6, siCTRL), 622 (USP37 KO6, siTRAIP), 806 (USP37 KO17, siCTRL), 476 (USP37 KO17, siTRAIP). Cells were analyzed from three biological replicates. Bars represent median. 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.

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.

Fig. 4. USP37 depletion induces TRAIP-dependent disassembly of CMGs converged at tandem DPCs, but not at terminated forks or ones separated by a LacR array.

a Top, a schematic of the DNA-protein cross-link substrate. meDPCs, methylated DNA-protein cross-links. The square bracket indicates the distance between distal leading-strand DPCs. Bottom, the meDPCs substrate was incubated in the indicated egg extracts in the presence or absence of p97-i. At specified times, chromatin was recovered and immunoblotted for the indicated proteins. Samples are from the same experiment; blots were processed in parallel. See also Supplementary Fig. 8a, b. b Same as (a), top, but the distance between distal meDPCs was 1033 bp. Bottom, the meDPCs substrate was incubated in the indicated egg extracts in the presence or absence of p97-i. Extracts were supplemented with recombinant WT TRAIP expressed in WGE or WGE with empty vector (EV). Samples are from the same experiment; blots were processed in parallel. Dashed blue line, probable CRL2^Lrr1-dependent ubiquitylation resulting from termination events. Dashed red line, TRAIP-dependent ubiquitylation. See also Supplementary Fig. 8e, f. c Top, schematic showing terminated CMGs and inhibited CRL2^Lrr1 by Cul-i. Bottom, plasmid DNA was replicated in the indicated egg extracts with p97-i and, where indicated, with Cul-i. At 90 min, chromatin was recovered and immunoblotted for the indicated proteins. See also Supplementary Fig. 9a, b. d Top, a schematic of CMGs converged at the meDPCs versus the LacR-bound lacO array. Bottom, plasmid DNA containing a 32x lacO array was preincubated with LacR and then replicated in the indicated egg extracts. At 30 min, reactions were supplemented with buffer or Cyclin B/CDK1. At the specified times, chromatin was recovered and immunoblotted for the indicated proteins. See also Supplementary Fig. 9e, f. e Same as (d), but at 40 min, reactions were supplemented with p97-i. See also Supplementary Fig. 9g, i. Source data are provided as a Source Data file.

Fig. 5. Predicted CDC45-USP37 interaction surface is important for USP37 function at the replisome.

a Xenopus sperm chromatin was incubated in egg extracts with indicated inhibitors for 10 min, recovered and blotted. See also Supplementary Fig. 10b. b Right, the predicted structure in Supplementary Fig. 10f aligned to the structure of human CMG (PDB:7pfo). Left, the same model rotated 90°. Orange dashed line, disordered region. c Xenopus sperm chromatin was incubated in egg extracts supplemented with WGE-expressed recombinant USP37 variants, recovered and blotted. Geminin was added where indicated to block replication. See also Supplementary Fig. 11a, b. d Plasmid DNA was incubated in the indicated egg extracts with different WGE-expressed recombinant USP37 variants, ICRF-193 and p97-i as indicated, recovered and blotted. Samples are from the same experiment, blots processed in parallel. See also Supplementary Fig. 11c, d. Abbreviations as in Supplementary Fig. 10k. e Extracts of HEK293T cells expressing mCherry (empty vector, EV), mCherry-USP37^WT, or mCherry-USP37^8A were subjected to mCherry immunoprecipitation (IP) followed by immunoblotting. n = 3 independent experiments. f Representative images of USP37-EdU PLA. Scale bar: 10 μm (left); Dot plot indicating the number of PLA foci between mCherry-tagged EV, USP37^WT, USP37^C350, or USP37^8A and EdU (replicating DNA) in untreated- or camptothecin-treated cells (right). Untreated: n = 291 (EV), 329 (WT), 307 (C350), 287 (8A) cells; camptothecin: n = 197 (EV), 320 (WT), 287 (C350), 300 (8A) cells. Bar, median of three independent experiments. Black dots, means in each biological replicates. Statistics reflect two-tailed Wilcoxon rank-sum tests for p values without adjustment for multiple hypothesis testing and Cohen’s d for effect sizes (γ). g, h Clonogenic survival assays of control (CTRL) or USP37 KO (clone 10) cells expressing mCherry (EV), mCherry-USP37^WT, or mCherry-USP37^8A treated with camptothecin (g) or etoposide (h). CTRL + EV and USP37 KO+ mCherry-USP37^WT samples are as in Fig. 1h, i. n = 3 independent experiments. Bars represent means ± SEM. Statistical analysis for (g, h) with two-way ANOVA and Šídák test for multiple comparisons. i Clonogenic survival assays of USP37 KO cells expressing mCherry (EV), mCherry-USP37^WT, or mCherry-USP37^8A transduced with a control or TRAIP-targeting sgRNA upon treatment with camptothecin. Bars, means ± SEM. Statistical analysis with two-way ANOVA and Holm-Šídák test for multiple comparisons. Source data provided as a Source Data file.

Fig. 6. Model of USP37’s role in protecting replisomes from premature disassembly.

In wild-type cells (WT), USP37 counteracts trans ubiquitylation by TRAIP (Supplementary Fig. 1a) when CMGs are stalled at sites of DPCs/topological stress. Thus, USP37 activity allows replisomes to eventually complete DNA synthesis and be unloaded by a termination-specific CRL2^Lrr1-dependent pathway. In the absence of USP37, TRAIP hyper-ubiquitylates CMG at sites of topological stress, causing premature CMG disassembly, DNA damage, and cell death. When both USP37 and TRAIP are absent converging CMGs cannot be prematurely ubiquitylated and, thus, may ultimately terminate normally. For simplicity, the region in between two stalled replisomes is shown as plectonemes.