USP37 prevents unscheduled replisome unloading through MCM complex deubiquitination

Abstract

The CMG helicase (CDC45-MCM2-7-GINS) unwinds DNA as a component of eukaryotic replisomes. Replisome (dis)assembly is tightly coordinated with cell cycle progression to ensure genome stability. However, factors that prevent premature CMG unloading and replisome disassembly are poorly described. Since disassembly is catalyzed by ubiquitination, deubiquitinases (DUBs) represent attractive candidates for safeguarding against untimely and deleterious CMG unloading. We combined a targeted loss-of-function screen with quantitative, single-cell analysis to identify human USP37 as a key DUB preventing replisome disassembly. We demonstrate that USP37 maintains active replisomes on S-phase chromatin and promotes normal cell cycle progression. Proteomics and enzyme assays revealed USP37 interacts with the CMG complex to deubiquitinate MCM7, thus antagonizing replisome disassembly. Significantly, USP37 protects normal epithelial cells from oncoprotein-induced replication stress. Our findings reveal USP37 to be critical to the maintenance of replisomes in S-phase and suggest USP37-targeting as a potential strategy for treating malignancies with defective DNA replication control.

Keywords: CMG; DNA replication; genome integrity; replisome; ubiquitin.

Figure 1.. A targeted siRNA screen identifies USP37 as the antagonizing DUB for replisome disassembly.

A. Model displaying the molecular players involved in replication termination. During replication termination, the CMG replicative helicase (CDC45-MCM2–7-GINS) is poly-ubiquitinated with lysine 48 (K48) ubiquitin linkages and the replisome is disassembled through p97. A deubiquitinase (DUB) could antagonize the ubiquitination-dependent disassembly to prevent premature replisome disassembly. B. Workflow for chromatin flow cytometry assays to study replication and bound MCM. RPE1-hTert cells were treated for 24 hours with either p97i or a panel of siRNAs to knock down selected DUBs individually (siDUB). Cells were labelled with EdU (thymidine analog) 30 minutes prior to harvesting, then soluble proteins were pre-extracted to retain only chromatin-bound proteins such as MCM2 (one of the replisome components). Cells were then fixed and stained for EdU (for active DNA synthesis), MCM2 (as a representative subunit for the MCM2–7 complex), and DAPI (for total DNA content) for flow cytometric analysis. C. Chromatin flow cytometry for RPE1-hTert cells treated with 20 nM siControl or 1.25 μM of CB-5083 (p97 inhibitor) for 24 hours, and pulsed with EdU for 30 min before harvesting. Cells were stained for bound MCM2, and DAPI (for DNA content). In the late S/G2/M gate, control cells are divided into high (>10³) versus low (<10³) bound MCM. Representative of two biological replicates. D. Histograms of the late S/G2/M-MCM^DNA-positive cells from (C). E. RPE1-hTert cells were treated with siControl or siDUB at 20 nM as indicated. Box and whisker plots for EdU intensity per cell in S phase. Cells in each sample were randomly down sampled to 2400 cells per sample. Data is combined from two independent biological replicates. Relative fold-change of the means of EdU intensity from the two replicates was computed: siControl versus siUSP37, unpaired two tailed t-test, p=0.0115. F. Bound MCM in late S/G2/M from cells treated as in (E). Left: Histograms of normalized counts of the late S/G2/M-MCM^DNA-positive cells. Right: Relative percentage of high MCM, late S/G2/M-MCM^DNA-positive cells from at least two independent biological replicates; mean with error bars ± SEM. Unpaired two tailed t-test for the means of the three replicates for siControl versus siUSP37, p<0.0001.

Figure 2.. USP37 prevents replisome disassembly in S phase

A. Illustration of CMG at active replisomes versus MCM loaded at unfired-origins that lack CDC45. B. Workflow. Cells were treated with siControl or siUSP37; doxycycline was added concurrently with the siRNA treatment to express the siRNA-resistant USP37. Cells were EdU-labelled and harvested after 24 hours and analyzed by flow cytometry for endogenous bound CDC45 and DNA synthesis. C. Immunoblotting for endogenous and ectopic USP37 in RPE1-hTert cells treated with siControl or siUSP37 at 5 nM ± doxycycline at 20 ng/mL. Representative of four biological replicates. D. Chromatin flow cytometry for the same samples in (B). Cells were stained for bound CDC45, EdU incorporation (for DNA synthesis) and DAPI (for DNA content). Data shown from S phase cells only (1500 cells in each plot). Representative of four biological replicates. E. Quantification of (D). Box and whisker plots for chromatin-bound CDC45 intensity per cell in S phase. The aggregate of four biological replicates was randomly down sampled to ~9600 cells per sample. Relative fold-change of the means of bound CDC45 intensity from the four replicates was computed: siControl versus siUSP37 or siUSP37 vs siUSP37 + USP37^R, unpaired two tailed t-test, p=0.0113, 0.0190, respectively. F. Box and whisker plots for EdU intensity per cell in S phase from cells treated as outlined in (B). The aggregate of three biological replicates was randomly down sampled to 7500 cells per sample. Relative fold-change of the means of EdU intensity from the three replicates was computed: siControl versus siUSP37 or siUSP37 vs siUSP37 + USP37^R, unpaired two tailed t-test, p=0.0748, 0.0455, respectively.

Figure 3.. USP37 interacts with the replisome components

A. Triplicate samples of HEK293T cells expressing FLAG-tagged USP37 or an empty vector (EV) control were subjected was FLAG immunoprecipitation. Immunoprecipitates were washed, eluted and subjected to proteomic analysis. Proteins enriched in USP37 IPs, compared to controls, are shown.

B. Gene Ontology analysis was performed on significantly enriched proteins to reveal the majority of USP37 interactors are involved in DNA replication and cell cycle progression. C. All components of the CMG complex were enriched in the FLAG-USP37 IP-MS. The fold enrichment over control, p-value, and number of peptides identified are shown. D. HEK293T cells were transfected for 48 hours with FLAG-tagged USP37 or an empty vector as a control. FLAG-USP37 was subjected to FLAG immunoprecipitation and analyzed by immunoblot. The indicated endogenous components of the CMG complex were co-precipitated by USP37. E. USP37 schematic. FL in (F) corresponds to full length USP37, D corresponds to USP37 lacking the PH domain, and PH corresponds to a USP37 fragment containing the PH domain only. F. The interaction between USP37 FL, Δ, or PH was assessed as described in D. USP37 interacts with the CMG complex through its PH domain.

Figure 4.. USP37 regulates the CMG complex by deubiquitinating MCM7

A. HEK293T cells were transfected for 48 hours with MCM7-V5, alone or in combination with FLAG-USP37. MCM7 interacts with USP37, as observed by immunoblot using the indicated antibodies. B. USP37 was knocked down using siRNA for 48 hours in RPE1 cells stably expressing a 6His-FLAG-tagged ubiquitin construct. Ubiquitinated proteins were pulled down using Ni-NTA, revealing that USP37 siRNA increases endogenous MCM7 ubiquitination, as observed by immunoblotting. C. MCM7 ubiquitination was analyzed as described in B, except that cells were treated with 5 μM of the p97i CB-5083 for the last 4 hours before harvesting. Inhibition of p97 strongly increases MCM7 ubiquitination, and this is even more pronounced after USP37 knock down. D. Ubiquitinated MCM7 isolated from HEK293T cells was mixed with 100 nM of recombinant USP37 WT or a catalytically inactive mutant (C350S). The in vitro deubiquitination assay shows that USP37 WT, but not C350S, deubiquitinates Ub-MCM7. E. Flag-tagged USP37 was ectopically expressed for 48 hours and subsequently purified from HEK-293T cells by FLAG immunoprecipitation. USP37 immunoprecipitates were mixed with 1 μM of K11, K48 or K63 tetra-ubiquitin chains, revealing that USP37 cleaves Tetra- and Tri-Ub more efficiently than Di-Ub.

Figure 5.. USP37 protects replication efficiency and proliferation in oncoprotein-expressing cells.

A. Expression-corrected CERES correlation scores were downloaded from The DepMap database for genes similar to USP37 knockout. Proteins involved in DNA replication and the DNA damage response are significantly enriched. Proteins are color coded similarly to Figure 3A (MCMs = blue, CDC45 = purple, polymerases = brown). B. RPE1-hTert cells engineered for either doxycycline-inducible Cyclin E1 or c-MYC were treated to induce expression simultaneously with USP37 depletion to examine effects on DNA replication and cellular fitness. C. Immunoblotting for USP37, Cyclin E1 or c-MYC in RPE1-hTert cells as outlined in B. siControl or siUSP37 were used at 5 nM. Doxycycline was added simultaneously with the siRNA at 100 or 25 ng/mL to overproduce Cyclin E1 or c-MYC for 24h, respectively as indicated. Representative of two biological replicates. D. EdU intensity per cell for the experiment described in (B) to overproduce Cyclin E1. The aggregate of two biological replicates was randomly down sampled to 2000 cells per sample. Relative fold-change of the means of EdU intensity from the two replicates: siControl versus siUSP37+ Cyclin E1 or siUSP37 versus siUSP37+ Cyclin E1 or Cyclin E1 versus siUSP37+ Cyclin E1, was computed, unpaired two tailed t-test, p=0.0009, 0.0006, 0.3059, respectively. E. Normalized fitness for RPE1-hTert Cyclin E1 cells treated with siControl or siUSP37 with or without overexpression of Cyclin E1 to induce replication stress for five days total. n = 3 biological replicates. * = p ≤ 0.05, ** = p ≤ 0.01 by one-way ANOVA. F. EdU intensity per cell for the experiment described in (B) to overproduce c-MYC. The aggregate of two biological replicates was randomly down sampled to 2000 cells per sample. Relative fold-change of the means of EdU intensity from the two replicates: siControl versus siUSP37+ c-MYC or siUSP37 versus siUSP37+ c-MYC or c-MYC versus siUSP37 + c-MYC, was computed, unpaired two tailed t-test, p=0.2044, 0.7278, 0.4, respectively. G. Normalized fitness for RPE1-hTert c-Myc cells treated with siCTRL or siUSP37 with or without overexpression of c-MYC to induce replication stress for three days total. n = 3 biological replicates. * = p ≤ 0.05, ** = p ≤ 0.01, **** = p ≤ 0.001 by one-way ANOVA.