IntroductionCells invest in extensive repair mechanisms to ensure fidelity of the genetic information stored in their DNA. Defective DNA repair results in mutagenesis and genome instability, major hallmarks of cancer, aging and aging-related diseases1,2. Cellular DNA repair activities are organized by sophisticated networks of post-translational modifications3,4. Regulatory ubiquitylation events are critical to recruit DNA repair factors in highly controlled manners. Mono-ubiquitylation of PCNA promotes DNA damage tolerance by recruiting translesion synthesis (TLS) polymerases5, while mono-ubiquitylation of the FANCD2/FANCI heterodimer traps the complex on DNA, initiating DNA repair by the Fanconi anemia pathway6.Tight regulation is especially important for DNA repair enzymes that are potentially toxic. The SPRTN protease employs a promiscuous activity to degrade covalent DNA-protein crosslinks (DPCs), but it has remained enigmatic how the enzyme achieves specificity for crosslinked proteins and how the unwanted cleavage of chromatin proteins is prevented. DPCs arise upon stabilization of covalent intermediates between DNA-processing enzymes and their substrates7. Additionally, various endogenous and environmental reactive agents crosslink proteins to DNA8,9. DPCs are toxic because they block DNA replication and transcription10,11,12,13. The collision of the replication machinery with crosslinked proteins initiates repair by SPRTN14,15, which can additionally be triggered by global-genome mechanisms9. The repair of DPCs by SPRTN is essential for viability. Its loss is lethal in human cell lines16 and leads to dramatic genome instability and early embryonic lethality in mice17.SPRTN features a metalloprotease domain at the N-terminus, which, together with the single-stranded DNA (ssDNA) -binding zinc-binding domain (ZBD), forms the conserved SprT domain (Fig. 1a)18,19. The SprT domain is followed by a basic region (BR) that interacts with double-stranded DNA (dsDNA)20. ZBD and BR couple SPRTN activity to the recognition of ssDNA-dsDNA junctions21, that arise when DNA polymerases stall at DPCs during replication14. However, the recognition of DNA junctions cannot explain how specificity is achieved during DPC repair, given that these structures are common throughout the genome, for example on the lagging strand during DNA replication. In addition to its DNA-binding domains, SPRTN bears interaction motifs for binding to the segregase p97 (SHP box) and PCNA (PIP box)22,23,24,25 but neither is required for SPRTN’s DPC repair function9,14,17. Furthermore, SPRTN carries a C-terminal ubiquitin-binding zinc finger (UBZ), promoting SPRTN ubiquitylation and thereby its inactivation26. A motif interacting with ubiquitin (MIU) has been predicted at SPRTN’s N-terminus but has not been experimentally confirmed27. The presence of ubiquitin-binding domains indicates a critical role of ubiquitin in regulating SPRTN-mediated DPC repair.Fig. 1: Ubiquitylation of DPCs promotes their cleavage by SPRTN.a Schematic of SPRTN’s domain structure and truncated variants, featuring motif interacting with ubiquitin (MIU), protease domain, zinc-binding domain (ZBD), basic region (BR), SHP box for p97-binding, PCNA-interacting motif (PIP) and ubiquitin-binding zinc finger (UBZ). SPRTNΔC is caused by a frameshift mutation resulting in a variant composed of SPRTN’s N-terminal 240 residues followed by eight additional amino acids (X8). b Schematic of HMCESSRAP ubiquitylation to generate DPCs shown in e, f, Fig. 4 and Supplementary Fig. 5b and 6b. HMCESSRAP-Ub(G76V)-3C-FKBP was incubated with FRB-E3 + E2 (K48 or K63) in the presence of ubiquitin, rapamycin, ubiquitin-E1 and ATP for 2 h (K63) or 6.5 h (K48) at 30 °C. After cleavage of the FKBP-tag via 3C-protease, ubiquitylated HMCESSRAP was purified by reverse immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography (SEC). c Mass spectrometry analysis of ubiquitin linkages formed by ubiquitylation of HMCESSRAP as shown in (b). Bar chart shows the mean ± SD of three biological replicates. d Schematic of the generation of HMCESSRAP-DPCs. HMCESSRAP was incubated for 30 min at 37 °C with a Cy5-labeled 30nt oligonucleotide containing a dU at position 15 and UDG. After crosslinking a complementary 15nt reverse oligonucleotide was annealed to form a ssDNA-dsDNA junction. e Indicated HMCESSRAP-DPCs (10 nM) were incubated alone or in the presence of FANCJ (100 nM) and indicated concentrations of SPRTN (1-100 nM) for 1 h at 30 °C. Quantification: bar graphs represent the mean ± SD of three independent experiments. All samples derive from the same experiment and gels were processed in parallel. Values for cleavage of unmodified HMCESSRAP-DPC are the same as in Supplementary Fig. 1b. Source data are provided as a Source Data file. f Indicated HMCESSRAP-DPCs (10 nM) were incubated alone or in the presence of FANCJ (100 nM) and indicated concentrations of SPRTN or SprT-BR (1-100 nM) for 1 h at 30 °C. Quantification: bar graphs represent the mean ± SD of three independent experiments. All samples derive from the same experiment and gels were processed in parallel. Source data are provided as a Source Data file.Full size imageIndeed, DPCs are ubiquitylated during replication by the ubiquitin-E3s TRAIP and RFWD314,15,28, while SUMOylation precedes ubiquitylation of the protein adduct by the SUMO-targeted ubiquitin-E3s RNF4 and TOPORS during global-genome repair9,29,30,31,32. DPC ubiquitylation can promote proteasomal degradation of crosslinked proteins9,14,15,29,30, but it has remained controversial whether it is important for SPRTN-mediated repair. Cleavage of a model DPC by SPRTN in frog egg extracts occurs even if the protein adduct has been treated with formaldehyde to prevent ubiquitylation14. Nonetheless, ubiquitylated DPCs accumulate upon SPRTN depletion33, indicating that they are substrates of the protease. Furthermore, SPRTN’s UBZ domain supports efficient DPC cleavage in frog egg extracts and cells9,14, which has led to the speculation that the UBZ may help to recruit SPRTN to ubiquitylated DPCs. Surprisingly however, the UBZ domain is not essential for SPRTN function. Patients with Ruijs-Aalfs syndrome (RJALS) express truncated versions of SPRTN that lack the C-terminal part of the enzyme including the UBZ (SPRTNΔC, Fig. 1a)27. RJALS patients suffer from premature aging and liver cancer27, phenotypes that are recapitulated in mice with reduced SPRTN function17. Yet, truncated SPRTN patient variants are clearly compatible with life, in contrast to full loss of SPRTN. Indeed, the severe growth defects associated with SPRTN loss in conditional mouse knock-out cells are rescued by expression of a truncated SPRTN variant34. It has remained enigmatic how SPRTN patient variants target DPCs in the absence of the UBZ and, more generally, whether and how SPRTN activity is regulated by DPC ubiquitylation.Here, we investigate the role of ubiquitin in SPRTN activation by biochemical reconstitution of DPC ubiquitylation, molecular dynamics (MD) simulations, NMR experiments and cellular assays. We find that DPC ubiquitylation activates SPRTN more than one hundred-fold. Activation occurs independently of SPRTN’s UBZ domain but involves a ubiquitin-binding interface at the back of its protease domain. This interface is required in cells expressing truncated RJALS patient variants to maintain genome stability and cellular fitness. Collectively, our results reveal a regulatory mechanism that confines SPRTN’s protease activity by linking its activation to DPC modification. Moreover, given that ubiquitin-dependent activation is retained in truncated SPRTN variants, our data explain how residual SPRTN function is maintained in RJALS patients.ResultsUbiquitylation of DNA-protein crosslinks promotes their cleavage by SPRTNTo directly test whether DPC ubiquitylation regulates SPRTN, we reconstituted DPC ubiquitylation in vitro. To modify DPCs with ubiquitin chains of defined linkages, we employed synthetic engineered ubiquitin-E3s (streamlined versions of the previously described Ubiquiton system35), enabling us to modify the catalytic SRAP domain of HMCES (HMCESSRAP) with K48- or K63-linked ubiquitin chains prior to DPC formation with an oligonucleotide containing an abasic (AP) site. HMCES actively crosslinks to AP sites within ssDNA to prevent AP site scission during DNA replication36. First, we fused a C-terminal tag containing a mono-ubiquitin moiety and a FK506-binding protein (FKBP) domain to HMCESSRAP. We then incubated this substrate with ubiquitin, an engineered ubiquitin-E3 carrying an FKBP-rapamycin-binding (FRB) domain, ubiquitin-E1, ubiquitin-E2, ATP and rapamycin (Fig. 1b). Rapamycin induces proximity between the substrate and the E3, promoting modification of the ubiquitin moiety fused to HMCESSRAP with either K48- or K63-linked polyubiquitin chains (depending on the identity of the E2/E3 enzymes used in the assay). Following cleavage of the 3C-site between ubiquitin and FKBP, HMCESSRAP modified with short or long ubiquitin chains was purified over several steps (Fig. 1b and Supplementary Fig. 1a, for all recombinant proteins used in this study). Mass spectrometry (MS) analysis confirmed the specific formation of K48- and K63-linked polyubiquitin chains on HMCESSRAP (Fig. 1c). DPCs were then generated by incubating unmodified or ubiquitylated HMCESSRAP with an AP site-containing fluorescently-labeled ssDNA-dsDNA junction (Fig. 1d)37,38.Next, we incubated the DPCs with SPRTN and the helicase FANCJ, which is required for SPRTN activity in these assays. FANCJ loads on the ssDNA portion of the substrate and translocates into the crosslinked protein, resulting in unfolding of the protein adduct, which in turn enables SPRTN to cleave the DPC37. SPRTN cleaved ubiquitylated DPCs more efficiently than unmodified protein adducts, with long chains activating stronger than shorter ones, independently of linkage type (Fig. 1e, lanes 7-16 (K48) and lanes 23-32 (K63)). The ubiquitin-dependent activation of SPRTN was substantial with the extent of cleavage of ubiquitylated DPCs by 1 nM of SPRTN being comparable to the cleavage of unmodified DPCs by 100 nM of SPRTN (Fig. 1e, compare lanes 5 and 13 (K48) and lanes 21 and 29 (K63)). Remarkably, in addition to the fragment produced upon cleavage of unmodified DPCs (Fig. 1e, Cleaved DPC), smaller cleavage products (Fig. 1e, Cleaved DPC*) appeared upon cleavage of ubiquitylated DPCs. Of note, smaller cleavage products were also detected upon addition of free K48- or K63-linked tetra-ubiquitin chains, although to a lesser extent (Supplementary Fig. 1b, cleaved DPC*, lanes 7-9 (K48) and lanes 17-19 (K63)).To test whether SPRTN’s known ubiquitin-binding domains are mediating the stimulating effect of DPC ubiquitylation, we utilized a minimal active SPRTN variant (SprT-BR, aa28-245), that lacks both, MIU and UBZ (Fig. 1a). While the truncated SprT-BR variant showed reduced cleavage of unmodified DPCs compared to the wild-type (WT) enzyme (Fig. 1f, compare lanes 3-5 with lanes 6-8), DPC ubiquitylation strongly boosted its activity (Fig. 1f, compare lanes 10-12 with lanes 13-15 (K48) and lanes 17-19 with lanes 20-22 (K63)). The stimulating effect of DPC ubiquitylation on truncated SprT-BR suggested to us that this region likely contains an additional ubiquitin-binding site that mediates the effect of ubiquitin on SPRTN activation.Ubiquitin promotes an open SPRTN conformationTo explore this possibility, we used ColabFold39 to predict complexes between SprT-BR and ubiquitin. In the top-ranked model, the hydrophobic Ile44-patch of ubiquitin was predicted to interact with a hydrophobic interface at the back of the SprT domain (Supplementary Fig. 2a-b), hereafter referred to as ubiquitin-binding interface at the SprT domain (USD). Interestingly, in all models, the SprT domain was predicted to adopt an open conformation with a highly accessible active site facing the DNA binding site of the ZBD. A similar conformation was also predicted in the absence of ubiquitin, in stark contrast to the published crystal structure of the SprT domain (PDB:6mdx19) that shows a closed conformation with the ZBD restricting access to the active site (Fig. 2a–c).Fig. 2: Ubiquitin promotes an open SPRTN conformation.a–c Experimental structure of SPRTN’s SprT domain (SPRTNaa28-214), PDB: 6mdx (a), ColabFold predicted structure of SprT (b) and ColabFold predicted structure of a SprT-ubiquitin (Ub1) complex (c). Protease domain is colored in blue, zinc-binding domain (ZBD) in orange and the Ub1 in grey. Zn2+ ions are colored in red. d–f Radius of gyration (Rg) of the indicated structures over 400 ns of molecular dynamics (MD) simulation. Each curve represents an independent MD trajectory (n = 3). Source data are provided as a Source Data file. g–i Main MD-clusters of the indicated structures during MD simulation for 400 ns, generated from three independent trajectories. For SprT (ColabFold predicted) two of three main MD-clusters are depicted. Rg correlating frequencies among all performed simulations are labeled above the structures. j, k Zoom-in to regions i and ii of the SprT-Ub1 complex (i), showing amino acids of ubiquitin (in grey) surrounding residue Leu38 (j) or L99 (k) of SPRTN (in blue) in the wild-type (WT) protein (left) and upon L38S or L99S replacement, respectively (right). l SprT-Ub1 binding energy difference (ΔΔG) between SprT-L38S or -L99S and WT protein obtained from alanine scanning. Bar graphs show the mean ± SD of 301 snapshots from PBSA calculations for the central structure of the largest cluster. Source data are provided as a Source Data file.Full size imageTo explore whether the predicted open SprT conformation is in equilibrium with the closed conformation and whether ubiquitin binding may affect SprT conformation, we conducted all-atoms MD simulations. We used either the crystal structure or ColabFold-based predictions of the SprT domain, alone or in combination with ubiquitin, as starting points (Fig. 2d–f and Supplementary Fig. 2c). The compact conformation observed in the crystal structure remained largely unchanged over the entire 400 ns timeframe in three independent simulations (Fig. 2d and Supplementary Movie 1). To reveal the predominant conformations within all simulations, we employed RMSD-based clustering (Fig. 2g-i), revealing a single cluster with a closed conformation (Fig. 2g). In contrast, simulations of the ColabFold-predicted SprT structure displayed larger conformational changes during the simulations (Fig. 2e). We observed collapses to a compact conformation with a smaller radius of gyration (Fig. 2e, red arrow). Collapses were followed by rapid reopening of the structure (Fig. 2e, dark blue trace) or retention of the compact conformation (Fig. 2e, light blue trace, and Supplementary Movie 2). Clustering revealed two clusters with an open conformation (Fig. 2h, left) and one cluster with a closed conformation (Fig. 2h, right). Strikingly, the presence of ubiquitin prevented transitions of the SprT domain to the closed conformation (Fig. 2f and Supplementary Movie 3) and simulations predominantly remained in an open conformation (Fig. 2i). Moreover, ubiquitin binding to the USD interface of the SprT remained stable across all three independent simulations (Fig. 2f). These data indicated to us that ubiquitin binding at the SprT domain may promote SPRTN activation by stabilizing an open conformation of the enzyme with an accessible active site.Next, we wanted to determine amino acid residues within the USD interface that are important for ubiquitin-binding. In the predicted SprT-ubiquitin complex, Leu38 and Leu99 of SPRTN appeared to mediate the interaction via hydrophobic interactions involving multiple amino acids within ubiquitin’s hydrophobic Ile44- and Ile36-patch, respectively (Fig. 2i-k and Supplementary Fig. 2d-e). Both residues, Leu38 and Leu99, are highly conserved throughout evolution (Supplementary Fig. 2f). To assess the effect of replacing either leucine residue with a hydrophilic serine (L38S, L99S), we conducted free energy end-point calculations using MMPBSA in conjunction with alanine scanning (see Methods for details), which enabled us to quantify the effect of each leucine-to-serine replacement to the overall binding affinity of the SprT-ubiquitin complex. We calculated a decrease in binding affinity of around 0.6 kcal/mol for the L38S replacement and a more substantial decrease of 3.74 kcal/mol for L99S (Fig. 2l). This effect is explained by replacement of Leu38 or Leu99 resulting in the loss of hydrophobic contacts to ubiquitin’s Ile44- and Ile36-patch, respectively (Fig. 2i–k and Supplementary Fig. 2d, e).Taken together, our MD simulations results suggest a model wherein ubiquitin binding to the USD promotes SPRTN activity by stabilizing an open conformation with an accessible active site.DNA- and ubiquitin-binding affect SPRTN’s conformation synergisticallyTo experimentally test whether ubiquitin binds to the USD interface and whether ubiquitin binding affects SPRTN’s interaction with DNA, we used NMR spectroscopy. Heteronuclear single quantum coherence (HSQC) spectra of SprT-BR showed well-dispersed peaks (Supplementary Fig. 3a, b). Comparisons with a ZBD-BR construct enabled us to transfer many chemical shifts based on our previous analysis of the ZBD-BR construct21 (Supplementary Fig. 3b, c, see Figure legend for details). In particular, we could unambiguously assign Trp ε1 1H,15N resonances to the ZBD (Fig. 3, zoom-ins, orange labels) and protease domain (Fig. 3, zoom-ins, blue labels). Next, we compared NMR spectra of SprT-BR and SprT-BR-L99S, which superimposed very well (Supplementary Fig. 3d), except for those resonances in vicinity to the mutation site, indicating that structural integrity is not affected upon replacement of Leu99. Upon adding ubiquitin in five-fold excess, we observed some changes in the protease domain of SprT-BR spectra (Fig. 3a, blue boxes). In the L99S variant, the effects of ubiquitin addition were reduced, implying that they correspond to ubiquitin binding to SPRTN’s USD interface (Fig. 3b, blue boxes). While the ubiquitin-induced effects were subtle and mostly affected resonances corresponding to the protease domain, we also observed line-broadening for signals corresponding to ZBD (Supplementary Fig. 3e, note Ile212). While Trp ε1 resonances were only marginally affected by the addition of ubiquitin (Fig. 3a, b, zoom-ins), the addition of an activating DNA structure in two-fold excess led to major spectral changes in ZBD-BR regions (Fig. 3c). DNA-induced line-broadening was comparable between WT and L99S constructs (Fig. 3d), demonstrating that alteration of the USD does not affect DNA binding. Strikingly, upon combined addition of both DNA and ubiquitin, severe line-broadening was observed in SprT-BR that was more pronounced than the individual effects of ubiquitin or DNA binding (Fig. 3e, red boxes), suggesting that the simultaneous binding of DNA and ubiquitin has synergistic effects on SPRTN’s conformation. These effects were virtually absent in the L99S variant (Fig. 3f, red boxes). Consistently, addition of ubiquitin with a mutated Ile44-patch had little effect (Supplementary Fig. 4a, b).Fig. 3: DNA- and ubiquitin-binding affect SPRTN’s conformation synergistically.a–f Comparison of NMR spectra, highlighting Trp ε1 amide signals in 1H,15N-HSQC experiments of SprT-BR and SprT-BR-L99S. Trp ε1 region is labeled and boxed (bottom). Resonance assignments corresponding to the Trp ε1’s in the zinc-binding domain (ZBD) are shown in orange and those in the protease domain in blue. Broadened or shifted signals upon dsDNA addition are shown as asterisk. a, b SprT-BR (a) and SprT-BR-L99S (b) alone (= Apo) (black), with mono-ubiquitin (Ub1) (5x molar excess) (red). Minor changes are boxed in blue to highlight the spectral differences between SprT-BR and SprT-BR-L99S upon adding Ub1. Zoom-in region in Supplementary Fig. 3e is marked with a black box (b). c, d SprT-BR (c) and SprT-BR-L99S (d) alone (black) (= Apo), with dsDNA (2x molar excess) (red). Some of the ZBD resonances affected by dsDNA are labeled in black while the unchanged are labeled in grey. e, f Superimpositions of SprT-BR (e) and SprT-BR-L99S (f) in the presence of dsDNA (2x molar excess) (black) and of both dsDNA (2x molar excess) and Ub1 (5x molar excess) (red). Additional resonance changes upon adding Ub1 to the dsDNA-bound SprT-BR are shown with red boxes.Full size imageCollectively, our NMR data indicate that ubiquitin amplifies the effects of DNA binding on SPRTN conformation allosterically by binding to the USD interface at the back of the protease domain. Interestingly, ubiquitin had only small effects on its own, implying that DNA binding occurs first and promotes ubiquitin binding at the USD.Ubiquitin stimulates DPC cleavage by binding to SPRTN’s USD interfaceTo test whether DPC ubiquitylation stimulates SPRTN activity through binding to the USD interface, we produced full-length SPRTN with an L38S or L99S substitution. Both variants showed cleavage of unmodified HMCESSRAP-DPCs to the same degree as the WT protein (Fig. 4a, compare lanes 3-5, with 6-8 (L38S) and 9-11 (L99S)). While DPC ubiquitylation increased overall activity also in USD mutant variants, the formation of smaller additional cleavage fragments (Cleaved DPC*) observed upon cleavage of ubiquitylated DPCs with the WT protease was reduced (L38S) or almost absent (L99S) (Fig. 4b, c, compare lanes 3-5 with lanes 6-8 (L38S) and lanes 9-11 (L99S)). Combination of the L38S and L99S substitution had no additional effects over the single L99S mutation (Supplementary Fig. 5a, b, compare lanes 6-8 (L99S) with lanes 9-11 (L38S + L99S)). These results suggest that DPC ubiquitylation promotes DPC cleavage through two distinct mechanisms. First, DPC ubiquitylation boosts overall cleavage by SPRTN independent of the USD interface (see Discussion). Second, DPC ubiquitylation allosterically activates SPRTN by binding to the USD interface, enabling the protease to cleave crosslinked proteins more efficiently.Fig. 4: The ubiquitin-dependent activation of SPRTN is mediated by the USD.a–c Indicated HMCESSRAP-DPCs (10 nM) were incubated alone or in the presence of FANCJ (100 nM) and indicated concentrations (0.1–100 nM) and variants of SPRTN (WT, L38S, L99S) for 1 h at 30 °C. Quantification: bar graphs represent the mean ± SD of three independent experiments. Source data are provided as a Source Data file.Full size imageSUMO-targeted DPC ubiquitylation activates SPRTN in vitro and in cellsEncouraged by the strong effects observed using the synthetic DPC ubiquitylation system, we wanted to reconstitute SUMO-targeted DPC ubiquitylation using the enzymes that modify crosslinked proteins in cells. Therefore, we generated DPCs using full-length HMCES protein (HMCESFL); we used HMCESFL because it contains a canonical SUMOylation site in its C-terminal tail that is absent in HMCESSRAP constructs. HMCESFL-DPCs were incubated with the SUMOylation machinery, consisting of SUMO-E1, SUMO-E2, SUMO-E3 PIAS4, SUMO2 and ATP (Fig. 5a, b). Successful SUMOylation of the crosslinked protein was indicated by slower migrating HMCESFL-DPC species that were absent in reactions lacking SUMO-E1 (Fig. 5b, compare lanes 3 and 4). For the subsequent ubiquitylation, SUMOylated DPCs were incubated with ubiquitin, ubiquitin-E1, ubiquitin-E2 UBE2D3 and the SUMO-targeted ubiquitin-E3 RNF4 (Fig. 5a, b). Ubiquitylation of SUMOylated DPCs was evident as further upshifts in gel migration and was confirmed by western blot (Fig. 5b, lane 7). We used MS to determine the identity of the ubiquitylated lysine residues and the involved ubiquitin linkages. We identified K48-, K63- and K11-linked ubiquitin chains on SUMOylated DPCs (Fig. 5c), as has been observed in cells32. Ubiquitin chains formed on various HMCES lysine residues and on three distinct SUMO2 lysine residues (Fig. 5d). Ubiquitylation was lost in the absence of ubiquitin-E1 or in the absence of SUMOylation (Fig. 5b, lanes 5 and 6 respectively), demonstrating bona fide SUMO-targeted DPC ubiquitylation.Fig. 5: SUMO-targeted DPC ubiquitylation activates SPRTN.a Schematic of SUMO-targeted ubiquitylation of HMCESFL-DPCs used in b-f and h. HMCESFL-DPCs were incubated alone or in the presence of SUMO2, UBC9 and PIAS4, with or without SAE1/UBA2 for 30 min at 37 °C. Next unmodified or SUMOylated HMCESFL-DPCs were incubated alone or in the presence of ubiquitin (Ub), RNF4, UBE2D3, with or without UBE1 for 30 min at 37 °C. b SUMO-targeted ubiquitylated HMCESFL-DPCs generated as described in (a), separated by denaturing SDS-PAGE and immunoblotting. Source data are provided as a Source Data file. c Mass spectrometry analysis of ubiquitin linkages formed by SUMO-targeted ubiquitylation of HMCESFL-DPCs. Bar chart shows the mean ± SD of four biological replicates. d Mass spectrometry analysis of lysine residues within HMCES or SUMO modified upon SUMO-targeted ubiquitylation. Violin blots show the mean ± SD of four biological replicates. e Indicated HMCESFL-DPCs (10 nM) were incubated alone or in the presence of FANCJ (100 nM) and SPRTN (100 nM) for 1 h at 30 °C. Quantifications: bar graphs represent the mean ± SD of three independent experiments. Source data are provided as a Source Data file. f Indicated HMCESFL-DPCs (10 nM) were incubated alone or in the presence of FANCJ (100 nM) and indicated concentrations (1-100 nM) and variants of SPRTN (WT, L38S, L99S) for 1 h at 30 °C. Quantifications: bar graphs represent the mean ± SD of three independent experiments. All samples derive from the same experiment and gels were processed in parallel. Source data are provided as a Source Data file. g HeLa-TREx SPRTNΔC Flp-In cells complemented with indicated YFP-SPRTNFL-Strep-tag variants were treated as depicted (top) with 5-azadC (10 µM) and harvested at indicated time points. DNMT1-DPCs were isolated using PxP (middle, see Methods) and analyzed by immunoblotting (bottom). Shown is a representative of three independent experiments. Source data are provided as a Source Data file. h Indicated HMCESFL-DPCs (10 nM) were incubated alone or in the presence of FANCJ (100 nM) and indicated concentrations (1–100 nM) and variants of SPRTN (FL-WT/L99S, ΔUBZ-WT/L99S, ΔC-WT/L99S) for 1 h at 30 °C. Quantifications: bar graphs represent the mean ± SD of three independent experiments. All samples derive from the same experiment and gels were processed in parallel. Source data are provided as a Source Data file.Full size imageNext, we incubated modified DPCs with SPRTN and FANCJ. Consistent with our results with the synthetic system, we observed enhanced cleavage of the ubiquitylated protein adduct by SPRTN, compared to unmodified DPCs and SUMOylated DPCs (Fig. 5e, compare lanes 3 and 5 with lane 7). Again, additional cleavage products appeared upon DPC ubiquitylation (Fig. 5e, Cleaved DPC*), which were reduced in variants with an altered USD interface (Fig. 5f, compare lanes 3-5 with lanes 6-8 (L38S) and lanes 9-11 (L99S)).To test whether SUMO-targeted DPC ubiquitylation activates SPRTN also in cells, we monitored the cleavage of DNA methyltransferase 1 (DNMT1)-DPCs induced with 5-azadC40. DNMT1-DPCs are swiftly SUMOylated41, triggering their ubiquitylation by RNF49,29,30 and TOPORS31,32 and, subsequently, cleavage by SPRTN. While SPRTNΔC cells are viable, they fail to efficiently cleave 5-azadC-induced DNMT1-DPCs9. Therefore, we complemented HeLa T-REx Flp-In cells expressing patient-mimicking SPRTNΔC alleles from the endogenous locus with doxycycline-inducible full length SPRTN variants (WT, E112Q, L38S and L99S) and assessed cleavage of DNMT1-DPCs by the purification of x-linked proteins (PxP) assay (refs. 9,42, Fig. 5g and Methods). DNMT1-DPCs formed in all cell lines upon 5-azadC treatment (Fig. 5g). Following a 2-h chase in drug-free media, a specific cleavage band formed in SPRTNΔC cells expressing SPRTN-WT but not in cells expressing catalytically inactive SPRTN-E112Q (Fig. 5g, red dots), as observed previously9 (DPCs are still resolved in these cells because they are additionally targeted by proteasomal degradation9,29). SPRTN-dependent DNMT1-DPC cleavage was strongly reduced in cells expressing SPRTN-L38S or SPRTN-L99S (Fig. 5g, red dots), indicating that SUMO-targeted ubiquitylation promotes DPC cleavage in cells by activating SPRTN at the USD interface.To corroborate this observation, we additionally assessed 5-azadC-induced SPRTN autocleavage (a marker of SPRTN activation) in the absence of DPC ubiquitylation. To abrogate ubiquitylation of DNMT1-DPCs, we depleted RNF4 using siRNA in HAP1 TOPORS knock-out cells. Simultaneous depletion of RNF4 and TOPORS resulted in a complete loss of SPRTN autocleavage (Supplementary Fig. 6a), confirming that DPC ubiquitylation is critical for efficient SPRTN activation in cells.Given that DNMT1-DPC repair in cells is compromised upon replacement of critical USD residues and upon loss of SPRTN’s C-terminal tail in RJALS SPRTNΔC patient variants9, we wanted to examine potential synergistic effects of both alterations using our reconstituted system. We compared cleavage of DPCs modified by SUMO-targeted ubiquitylation by SPRTNFL and SPRTNΔC with intact or mutated USD interfaces. While SPRTNΔC displayed only slightly reduced DPC cleavage compared to the WT enzyme (Fig. 5h, compare lanes 3-5 with lanes 9-11), the extent of cleavage by SPRTNΔC was strongly reduced upon additional replacement of Leu99 by serine (Fig. 5h, compare lanes 9-11 and lanes 18-20). The synthetic cleavage defect of SPRTNΔC-L99S was only partially explained by loss of the UBZ domain, given that SPRTNΔUBZ-L99S variant displayed a less pronounced phenotype (Fig. 5h, lanes 15–17). Notably, the defect of SPRTNΔC was specific to DPCs modified by SUMO-targeted ubiquitylation. DPCs modified using the synthetic ubiquitylation system were cleaved comparably well by SPRTNΔC and the WT enzyme, while a USD mutant variant (L99S) displayed clear defects (Supplementary Fig. 6b and Discussion).Taken together, our results suggest that SUMO-targeted DPC ubiquitylation allosterically activates SPRTN at the USD interface to promote DPC repair. Our in vitro data further imply that the ubiquitin-dependent activation of SPRTN is specifically important to support the residual cleavage of RJALS SPRTNΔC patient variants towards DPCs modified by SUMO-targeted ubiquitylation.Ubiquitin-dependent activation of SPRTN maintains genome stability in Ruijs-Aalfs syndromeNext, we wanted to determine whether the ubiquitin-dependent activation of SPRTN at the USD interface is important to maintain the residual function of SPRTNΔC patient variants in cells. To this end, we complemented conditional SprtnF/-CreERT2 knock-out mouse embryonic fibroblasts (MEFs) with either an empty vector (EV) or different human SPRTN variants (FL and ΔC) tagged with a C-terminal Strep-tag (Supplementary Fig. 7a, b). Of note, SPRTNΔC variants expressed at much higher levels than the WT enzyme (Supplementary Fig. 7a, b), as previously observed in RJALS patients27. Loss of endogenous Sprtn was induced by 4-hydroxytamoxifen (4-OHT), with the solvent MeOH serving as control (Supplementary Fig. 7c, d), and resulted in diverse phenotypes including growth arrest (Fig. 6a, b), formation of micronuclei and chromatin bridges (Fig. 6c–e), as wells as arrest in the G2/M phase of the cell cycle (Supplementary Fig. 7e–h), as described previously17. All phenotypes were rescued by expression of human WT SPRTN but not by catalytically inactive SPRTN-E112Q (Fig. 6a and d). Also, expression of SPRTNΔC complemented all phenotypes induced by Sprtn knock-out (Fig. 6b and e). While the replacement of USD residues Leu38 or Leu99 had no effect on the ability of full-length SPRTN to complement cell fitness and cell cycle defects upon loss of mouse Sprtn (Fig. 6a and Supplementary Fig. 7e), loss of Leu99 resulted in intermediate growth defects and G2/M arrest in SPRTNΔC (Fig. 6b and Supplementary Fig. 7f). These defects were accompanied by severe signs of genome instability, observed as micronuclei and chromatin bridges in cells expressing SPRTNΔC-L99S (Fig. 6c and e).Fig. 6: Ubiquitin-dependent activation of SPRTN maintains genome stability in Ruijs-Aalfs syndrome.a, b Proliferation of SprtnF/- Cre-ERT2 mouse embryonic fibroblasts (MEFs) complemented with indicated SPRTN variants or empty vector (EV, pMSCV) treated with methanol (MeOH) or (Z)−4-hydroxytamoxifen (4-OHT) (2 µM) for 48 h. After seeding, cell numbers were counted at indicated time points. Values are the mean ± SD of eight technical replicates. Shown is a representative of three independent experiments. Source data are provided as a Source Data file. c Image showing micronuclei (asteriks) and chromatin bridges (arrow) in SprtnF/- Cre-ERT2 MEFs + pMSCV-SPRTNΔC-L99S treated with 4-OHT (2 µM) for 48 h. DNA was visualized by DAPI staining. Scale bar corresponds to 15 µm. d, e Quantification of micronuclei and chromatin bridges formation in SprtnF/- Cre-ERT2 MEFs complemented with indicated SPRTN variants or EV (pMSCV) treated with MeOH or 4-OHT (2 µM) for 48 h. DNA was visualized by DAPI staining. Bar graphs show the mean ± SD of three independent experiments. The p values were calculated using a two-way ANOVA with Dunnett’s multiple comparison test. P values: d Micronuclei (left): SPRTN-WT vs. SPRTN-L38S = 0.0002; SPRTN-WT vs. SPRTN-L99S Article CAS Google Scholar Miller, B. R. I. et al. MMPBSA.py: An Efficient Program for End-State Free Energy Calculations. J. Chem. Theory Comput. 8, 3314–3321 (2012).Article CAS PubMed Google Scholar Valdés-Tresanco, M. S., Valdés-Tresanco, M. E., Valiente, P. A. & Moreno, E. gmx_MMPBSA: A New Tool to Perform End-State Free Energy Calculations with GROMACS. J. Chem. Theory Comput. 17, 6281–6291 (2021).Article PubMed Google Scholar Nguyen, H., Roe, D. R. & Simmerling, C. Improved Generalized Born Solvent Model Parameters for Protein Simulations. J. Chem. Theory Comput. 9, 2020–2034 (2013).Article CAS PubMed PubMed Central Google Scholar Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).Article CAS PubMed Google Scholar Lee, W., Tonelli, M. & Markley, J. L. NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy. Bioinformatics 31, 1325–1327 (2015).Article PubMed Google Scholar Mulder, F. A., Schipper, D., Bott, R. & Boelens, R. Altered flexibility in the substrate-binding site of related native and engineered high-alkaline Bacillus subtilisins. J. Mol. Biol. 292, 111–123 (1999).Article CAS PubMed Google Scholar Download referencesAcknowledgementsWe thank B. Schulman for providing a plasmid for ubiquitin. We gratefully acknowledge Jiaxuan Chen from the IMB Proteomics Core Facility in Mainz for help with mass spectrometry experiments. S.D. and P.W. are supported by the International Max-Planck Research School for Molecules of Life. We are grateful to Sam Asami and Gerd Gemmecker for help with NMR measurements at the Bavarian NMR center. We thank Dr. Shar-yin N. Huang at the National Cancer Institute for her technical support. Research in the lab of J.S. is funded by European Research Council (ERC StG 801750 DNAProteinCrosslinks, ERC CoG 101124695 DECONSTRUCT), the Alfried-Krupp von Bohlen und Halbach-Stiftung, European Molecular Biology Organization (YIP4644), a Vallee Foundation Scholarship, and Deutsche Forschungsgemeinschaft (Project ID 213249687 - SFB 1064). H.D.U. acknowledges funding by the European Research Council (ERC AdG 101140447). We acknowledge funding by the Deutsche Forschungsgemeinschaft (J.S., H.D.U., and P.B.: Project-ID 393547839 – SFB 1361; M.S. and L.J.D: Project-ID 325871075 – SFB1309). The authors acknowledge the scientific support and HPC resources provided by the Erlangen National High Performance Computing Center (NHR@FAU) of the Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU) under the NHR project b119ee and the resources on the LiCCA HPC cluster of the University of Augsburg, co-funded by the Deutsche Forschungsgemeinschaft under Project-ID 499211671).FundingOpen Access funding enabled and organized by Projekt DEAL.Author informationAuthors and AffiliationsGene Center, Ludwig-Maximilians-Universität München, Munich, GermanySophie Dürauer, Dina S. Schnapka, Denitsa Yaneva, Maximilian J. Götz, Pedro Weickert & Julian StingeleDepartment of Biochemistry, Ludwig-Maximilians-Universität München, Munich, GermanySophie Dürauer, Dina S. Schnapka, Denitsa Yaneva, Maximilian J. Götz, Pedro Weickert & Julian StingeleInstitute of Structural Biology, Molecular Targets and Therapeutics Center, Helmholtz Munich, Neuherberg, GermanyHyun-Seo Kang & Michael SattlerBavarian NMR Center and Department of Bioscience, TUM School of Natural Sciences, Technical University of Munich, Garching, GermanyHyun-Seo Kang & Michael SattlerInstitute of Physics, University of Augsburg, Augsburg, GermanyChristian Wiebeler, Abigail C. Major & Nadine SchwierzDevelopmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USAYuka Machida & Yuichi J. MachidaInstitute of Molecular Biology gGmbH, Mainz, GermanyChristian Renz, Aldwin S. Rahmanto, Petra Beli & Helle D. UlrichInstitute of Developmental Biology and Neurobiology (IDN), Johannes Gutenberg-Universität Mainz, Mainz, GermanyAldwin S. Rahmanto & Petra BeliChair of Bioinorganic Chemistry, Heinrich-Heine Universität Düsseldorf, Düsseldorf, GermanySophie M. Gutenthaler-Tietze & Lena J. DaumannDepartment of Chemistry, Ludwig-Maximilians-Universität München, Munich, GermanySophie M. Gutenthaler-TietzeAuthorsSophie DürauerView author publicationsSearch author on:PubMed Google ScholarHyun-Seo KangView author publicationsSearch author on:PubMed Google ScholarChristian WiebelerView author publicationsSearch author on:PubMed Google ScholarYuka MachidaView author publicationsSearch author on:PubMed Google ScholarDina S. SchnapkaView author publicationsSearch author on:PubMed Google ScholarDenitsa YanevaView author publicationsSearch author on:PubMed Google ScholarChristian RenzView author publicationsSearch author on:PubMed Google ScholarMaximilian J. GötzView author publicationsSearch author on:PubMed Google ScholarPedro WeickertView author publicationsSearch author on:PubMed Google ScholarAbigail C. MajorView author publicationsSearch author on:PubMed Google ScholarAldwin S. RahmantoView author publicationsSearch author on:PubMed Google ScholarSophie M. Gutenthaler-TietzeView author publicationsSearch author on:PubMed Google ScholarLena J. DaumannView author publicationsSearch author on:PubMed Google ScholarPetra BeliView author publicationsSearch author on:PubMed Google ScholarHelle D. UlrichView author publicationsSearch author on:PubMed Google ScholarMichael SattlerView author publicationsSearch author on:PubMed Google ScholarYuichi J. MachidaView author publicationsSearch author on:PubMed Google ScholarNadine SchwierzView author publicationsSearch author on:PubMed Google ScholarJulian StingeleView author publicationsSearch author on:PubMed Google ScholarContributionsConceptualization: S.D. and J.S. Investigation: S.D., D.S.S., D.Y., P.W., M.J.G., Y.M., Y.J.M., and C.R. NMR: H.S.K. MD-simulations: C.W., A.C.M., and N.S. Mass spectrometry: A.S.R. ICO-OES: S.M.G.T. Writing – Original draft: S.D. and J.S. Writing – Review & Editing: S.D. and J.S. with input from all authors. Funding Acquisition and Supervision: J.S., L.J.D., P.B., H.D.U., M.S., Y.J.M., and N.S.Corresponding authorCorrespondence to Julian Stingele.Ethics declarationsCompeting interestsThe authors declare no competing interests.Peer reviewPeer review informationNature Communications thanks Javier O. Sanlley Hernandez and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. 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