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USP11 mediates repair of DNA–protein cross-links by deubiquitinating SPRTN metalloprotease

2021; Elsevier BV; Volume: 296; Linguagem: Inglês

10.1016/j.jbc.2021.100396

ISSN

1083-351X

Autores

Megan C. Perry, Meghan Biegert, Sai Sundeep Kollala, Halle J. Mallard, Grace Su, Manohar Kodavati, Natasha Kreiling, Alexander Holbrook, Gargi Ghosal,

Tópico(s)

Cancer-related Molecular Pathways

Resumo

DNA–protein cross-links (DPCs) are toxic DNA lesions that interfere with DNA metabolic processes such as replication, transcription, and recombination. USP11 deubiquitinase participates in DNA repair, but the role of USP11 in DPC repair is not known. SPRTN is a replication-coupled DNA-dependent metalloprotease that cleaves proteins cross-linked to DNA to promote DPC repair. SPRTN function is tightly regulated by a monoubiquitin switch that controls SPRTN auto-proteolysis and chromatin accessibility during DPC repair. Previously, VCPIP1 and USP7 deubiquitinases have been shown to regulate SPRTN. Here, we identify USP11 as an SPRTN deubiquitinase. USP11 interacts with SPRTN and cleaves monoubiquitinated SPRTN in cells and in vitro. USP11 depletion impairs SPRTN deubiquitination and promotes SPRTN auto-proteolysis in response to formaldehyde-induced DPCs. Loss of USP11 causes an accumulation of unrepaired DPCs and cellular hypersensitivity to treatment with DPC-inducing agents. Our findings show that USP11 regulates SPRTN auto-proteolysis and SPRTN-mediated DPC repair to maintain genome stability. DNA–protein cross-links (DPCs) are toxic DNA lesions that interfere with DNA metabolic processes such as replication, transcription, and recombination. USP11 deubiquitinase participates in DNA repair, but the role of USP11 in DPC repair is not known. SPRTN is a replication-coupled DNA-dependent metalloprotease that cleaves proteins cross-linked to DNA to promote DPC repair. SPRTN function is tightly regulated by a monoubiquitin switch that controls SPRTN auto-proteolysis and chromatin accessibility during DPC repair. Previously, VCPIP1 and USP7 deubiquitinases have been shown to regulate SPRTN. Here, we identify USP11 as an SPRTN deubiquitinase. USP11 interacts with SPRTN and cleaves monoubiquitinated SPRTN in cells and in vitro. USP11 depletion impairs SPRTN deubiquitination and promotes SPRTN auto-proteolysis in response to formaldehyde-induced DPCs. Loss of USP11 causes an accumulation of unrepaired DPCs and cellular hypersensitivity to treatment with DPC-inducing agents. Our findings show that USP11 regulates SPRTN auto-proteolysis and SPRTN-mediated DPC repair to maintain genome stability. DNA–protein cross-links (DPCs) are irreversible covalent cross-linking of proteins to the DNA. DPCs can be generated by the action of oxygen free radicals, reactive nitrogen species, and reactive aldehydes generated as by-products of cellular respiration and metabolism or by exposure to exogenous DNA damaging agents such as UV radiation, ionizing radiation (IR), and chemotherapeutic drugs (1Barker S. Weinfeld M. Murray D. DNA-protein crosslinks: Their induction, repair, and biological consequences.Mutat. Res. 2005; 589: 111-135Crossref PubMed Scopus (227) Google Scholar, 2Tretyakova N.Y. Groehler A.t. Ji S. DNA-protein cross-links: Formation, structural identities, and biological outcomes.Acc. Chem. Res. 2015; 48: 1631-1644Crossref PubMed Scopus (87) Google Scholar). Covalent cross-linking of proteins to the unperturbed duplex DNA, generated by formaldehyde, IR, UV rays, and platinum-based chemotherapeutic drugs, is classified as type 1 DPCs. Trapped DNA Polβ and PARP1 at the 5′ and 3′ ends of single-stranded DNA breaks (SSBs), respectively, represent type 2 DPCs. Type 3 and type 4 DPCs arise from abortive topoisomerase–DNA enzymatic reactions, resulting in the cross-linking of topoisomerase I (TOP1) to the 3′ end of an SSB or topoisomerase II (TOP2) to the two 5′ ends of a double-stranded DNA break (DSB) (3Ide H. Nakano T. Salem A.M.H. Shoulkamy M.I. DNA-protein cross-links: Formidable challenges to maintaining genome integrity.DNA Repair (Amst.). 2018; 71: 190-197Crossref PubMed Scopus (20) Google Scholar). Cross-linking of specific DNA metabolizing enzymes, such as TOP1 and TOP2, DNA polymerase, and DNA methyl transferase (DNMT1), is also known as enzymatic DPCs (4Stingele J. Bellelli R. Boulton S.J. Mechanisms of DNA-protein crosslink repair.Nat. Rev. Mol. Cell Biol. 2017; 18: 563-573Crossref PubMed Scopus (99) Google Scholar). Irrespective of the type or source of the lesion, all DPCs are steric blockades that disrupt DNA replication, transcription, recombination, and repair processes. Unrepaired or misrepaired DPCs lead to genome instability, resulting in tumorigenesis and genetic diseases (4Stingele J. Bellelli R. Boulton S.J. Mechanisms of DNA-protein crosslink repair.Nat. Rev. Mol. Cell Biol. 2017; 18: 563-573Crossref PubMed Scopus (99) Google Scholar, 5Fielden J. Ruggiano A. Popovic M. Ramadan K. DNA protein crosslink proteolysis repair: From yeast to premature ageing and cancer in humans.DNA Repair (Amst.). 2018; 71: 198-204Crossref PubMed Scopus (24) Google Scholar). Given the different types of DPCs and myriad of agents that generate them, the precise molecular mechanism underlying the DPC repair pathway has remained elusive. Genetic and biochemical experiments in different model organisms have suggested the nucleotide excision repair (NER) and homologous recombination (HR) pathways mitigate the genotoxic effects of DPCs (6Nakano T. Morishita S. Katafuchi A. Matsubara M. Horikawa Y. Terato H. Salem A.M. Izumi S. Pack S.P. Makino K. Ide H. Nucleotide excision repair and homologous recombination systems commit differentially to the repair of DNA-protein crosslinks.Mol. Cell. 2007; 28: 147-158Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 7Ide H. Shoulkamy M.I. Nakano T. Miyamoto-Matsubara M. Salem A.M. Repair and biochemical effects of DNA-protein crosslinks.Mutat. Res. 2011; 711: 113-122Crossref PubMed Scopus (80) Google Scholar, 8de Graaf B. Clore A. McCullough A.K. Cellular pathways for DNA repair and damage tolerance of formaldehyde-induced DNA-protein crosslinks.DNA Repair (Amst.). 2009; 8: 1207-1214Crossref PubMed Scopus (74) Google Scholar, 9Baker D.J. Wuenschell G. Xia L. Termini J. Bates S.E. Riggs A.D. O'Connor T.R. Nucleotide excision repair eliminates unique DNA-protein cross-links from mammalian cells.J. Biol. Chem. 2007; 282: 22592-22604Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The repair of TOP1 cleavage complexes (TOP1-ccs) and TOP2 cleavage complexes (TOP2-ccs) has been widely investigated. TOP1-ccs and TOP2-ccs are enzymatic DPCs repaired by tyrosyl-DNA phosphodiesterases TDP1 and TDP2, respectively. TDP1 and TDP2 directly hydrolyze the phosphotyrosyl bond between the protein and DNA in the DPC. Following DPC removal, the DNA breaks are subsequently repaired by SSB, HR, or nonhomologous end joining repair pathways (3Ide H. Nakano T. Salem A.M.H. Shoulkamy M.I. DNA-protein cross-links: Formidable challenges to maintaining genome integrity.DNA Repair (Amst.). 2018; 71: 190-197Crossref PubMed Scopus (20) Google Scholar, 10Pommier Y. Huang S.Y. Gao R. Das B.B. Murai J. Marchand C. Tyrosyl-DNA-phosphodiesterases (TDP1 and TDP2).DNA Repair (Amst.). 2014; 19: 114-129Crossref PubMed Scopus (169) Google Scholar). In addition to TDP1, APEX2 in human and Apn2 in yeast have been shown to remove TOP1-ccs (11Li F. Wang Q. Seol J.H. Che J. Lu X. Shim E.Y. Lee S.E. Niu H. Apn2 resolves blocked 3' ends and suppresses Top1-induced mutagenesis at genomic rNMP sites.Nat. Struct. Mol. Biol. 2019; 26: 155-163Crossref PubMed Scopus (18) Google Scholar, 12Alvarez-Quilon A. Wojtaszek J.L. Mathieu M.C. Patel T. Appel C.D. Hustedt N. Rossi S.E. Wallace B.D. Setiaputra D. Adam S. Ohashi Y. Melo H. Cho T. Gervais C. Munoz I.M. et al.Endogenous DNA 3' blocks are vulnerabilities for BRCA1 and BRCA2 deficiency and are reversed by the APE2 nuclease.Mol. Cell. 2020; 78: 1152-1165.e1158Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). The proteasome also aids the removal of TOP1-ccs, TOP2-ccs, Polβ cross-linked to DNA, and DPCs generated by formaldehyde. However, polyubiquitination of DPCs was observed only in TOP1-ccs and TOP2-ccs, not in formaldehyde-induced DPCs (13Nakano T. Katafuchi A. Matsubara M. Terato H. Tsuboi T. Masuda T. Tatsumoto T. Pack S.P. Makino K. Croteau D.L. Van Houten B. Iijima K. Tauchi H. Ide H. Homologous recombination but not nucleotide excision repair plays a pivotal role in tolerance of DNA-protein cross-links in mammalian cells.J. Biol. Chem. 2009; 284: 27065-27076Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 14Quievryn G. Zhitkovich A. Loss of DNA-protein crosslinks from formaldehyde-exposed cells occurs through spontaneous hydrolysis and an active repair process linked to proteosome function.Carcinogenesis. 2000; 21: 1573-1580Crossref PubMed Google Scholar). Studies in Xenopus egg extracts showed that when the replisome collides with DPCs, the CMG helicase stalls and the DPC is proteolyzed into a peptide–DNA adduct that is bypassed by translesion synthesis (TLS) polymerases, but proteasome inhibition had no significant effect on DPC repair (15Duxin J.P. Dewar J.M. Yardimci H. Walter J.C. Repair of a DNA-protein crosslink by replication-coupled proteolysis.Cell. 2014; 159: 346-357Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Concurrently, yeast Wss1 was identified as a DNA-dependent metalloprotease that cleaves both enzymatic TOP1-ccs and nonenzymatic formaldehyde-induced DPCs during S-phase (16Stingele J. Schwarz M.S. Bloemeke N. Wolf P.G. Jentsch S. A DNA-dependent protease involved in DNA-protein crosslink repair.Cell. 2014; 158: 327-338Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Subsequently, SPRTN protease was shown to repair DPCs in mammalian cells (17Stingele J. Bellelli R. Alte F. Hewitt G. Sarek G. Maslen S.L. Tsutakawa S.E. Borg A. Kjaer S. Tainer J.A. Skehel J.M. Groll M. Boulton S.J. Mechanism and regulation of DNA-protein crosslink repair by the DNA-dependent metalloprotease SPRTN.Mol. Cell. 2016; 64: 688-703Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 18Vaz B. Popovic M. Newman J.A. Fielden J. Aitkenhead H. Halder S. Singh A.N. Vendrell I. Fischer R. Torrecilla I. Drobnitzky N. Freire R. Amor D.J. Lockhart P.J. Kessler B.M. et al.Metalloprotease SPRTN/DVC1 orchestrates replication-coupled DNA-protein crosslink repair.Mol. Cell. 2016; 64: 704-719Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 19Lopez-Mosqueda J. Maddi K. Prgomet S. Kalayil S. Marinovic-Terzic I. Terzic J. Dikic I. SPRTN is a mammalian DNA-binding metalloprotease that resolves DNA-protein crosslinks.Elife. 2016; 5e21491Crossref PubMed Scopus (65) Google Scholar). Recently, Ddi1 aspartic protease was identified in yeast and was shown to repair DPCs independent of the 20S proteasome (20Serbyn N. Noireterre A. Bagdiul I. Plank M. Michel A.H. Loewith R. Kornmann B. Stutz F. The aspartic protease Ddi1 contributes to DNA-protein crosslink repair in yeast.Mol. Cell. 2019; 77: 1066-1079.e9Abstract Full Text Full Text PDF Scopus (20) Google Scholar). Collectively, these studies suggest that DPCs are degraded and removed by a repair pathway that is dependent on either the proteasome or a specific protease. SPRTN (also known as DVC1/C1orf124), the mammalian functional homolog of yeast Wss1, is a replication-coupled DNA-dependent metalloprotease (17Stingele J. Bellelli R. Alte F. Hewitt G. Sarek G. Maslen S.L. Tsutakawa S.E. Borg A. Kjaer S. Tainer J.A. Skehel J.M. Groll M. Boulton S.J. Mechanism and regulation of DNA-protein crosslink repair by the DNA-dependent metalloprotease SPRTN.Mol. Cell. 2016; 64: 688-703Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 18Vaz B. Popovic M. Newman J.A. Fielden J. Aitkenhead H. Halder S. Singh A.N. Vendrell I. Fischer R. Torrecilla I. Drobnitzky N. Freire R. Amor D.J. Lockhart P.J. Kessler B.M. et al.Metalloprotease SPRTN/DVC1 orchestrates replication-coupled DNA-protein crosslink repair.Mol. Cell. 2016; 64: 704-719Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). SPRTN was initially identified as a protein required for repair of UV-induced DNA lesions, restart of stalled DNA replication forks, and as a regulator of TLS (21Ghosal G. Leung J.W. Nair B.C. Fong K.W. Chen J. Proliferating cell nuclear antigen (PCNA)-binding protein C1orf124 is a regulator of translesion synthesis.J. Biol. Chem. 2012; 287: 34225-34233Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 22Centore R.C. Yazinski S.A. Tse A. Zou L. Spartan/C1orf124, a reader of PCNA ubiquitylation and a regulator of UV-induced DNA damage response.Mol. Cell. 2012; 46: 625-635Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 23Mosbech A. Gibbs-Seymour I. Kagias K. Thorslund T. Beli P. Povlsen L. Nielsen S.V. Smedegaard S. Sedgwick G. Lukas C. Hartmann-Petersen R. Lukas J. Choudhary C. Pocock R. Bekker-Jensen S. et al.DVC1 (C1orf124) is a DNA damage-targeting p97 adaptor that promotes ubiquitin-dependent responses to replication blocks.Nat. Struct. Mol. Biol. 2012; 19: 1084-1092Crossref PubMed Scopus (123) Google Scholar, 24Juhasz S. Balogh D. Hajdu I. Burkovics P. Villamil M.A. Zhuang Z. Haracska L. Characterization of human Spartan/C1orf124, an ubiquitin-PCNA interacting regulator of DNA damage tolerance.Nucleic Acids Res. 2012; 40: 10795-10808Crossref PubMed Scopus (70) Google Scholar, 25Davis E.J. Lachaud C. Appleton P. Macartney T.J. Nathke I. Rouse J. DVC1 (C1orf124) recruits the p97 protein segregase to sites of DNA damage.Nat. Struct. Mol. Biol. 2012; 19: 1093-1100Crossref PubMed Scopus (103) Google Scholar, 26Machida Y. Kim M.S. Machida Y.J. Spartan/C1orf124 is important to prevent UV-induced mutagenesis.Cell Cycle. 2012; 11: 3395-3402Crossref PubMed Scopus (54) Google Scholar, 27Kim M.S. Machida Y. Vashisht A.A. Wohlschlegel J.A. Pang Y.P. Machida Y.J. Regulation of error-prone translesion synthesis by Spartan/C1orf124.Nucleic Acids Res. 2013; 41: 1661-1668Crossref PubMed Scopus (43) Google Scholar). SPRTN associates with the DNA replication machinery and loss of SPRTN impaired replication fork progression (18Vaz B. Popovic M. Newman J.A. Fielden J. Aitkenhead H. Halder S. Singh A.N. Vendrell I. Fischer R. Torrecilla I. Drobnitzky N. Freire R. Amor D.J. Lockhart P.J. Kessler B.M. et al.Metalloprotease SPRTN/DVC1 orchestrates replication-coupled DNA-protein crosslink repair.Mol. Cell. 2016; 64: 704-719Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). SPRTN protease activity is mediated by the SprT domain of SPRTN, which contains the HEXXH catalytic motif. Like Wss1, SPRTN protease cleaves TOP1, TOP2, histone H1, H2A, H2B, H3, and HMG1 in the presence of single-stranded DNA (ssDNA) (17Stingele J. Bellelli R. Alte F. Hewitt G. Sarek G. Maslen S.L. Tsutakawa S.E. Borg A. Kjaer S. Tainer J.A. Skehel J.M. Groll M. Boulton S.J. Mechanism and regulation of DNA-protein crosslink repair by the DNA-dependent metalloprotease SPRTN.Mol. Cell. 2016; 64: 688-703Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 18Vaz B. Popovic M. Newman J.A. Fielden J. Aitkenhead H. Halder S. Singh A.N. Vendrell I. Fischer R. Torrecilla I. Drobnitzky N. Freire R. Amor D.J. Lockhart P.J. Kessler B.M. et al.Metalloprotease SPRTN/DVC1 orchestrates replication-coupled DNA-protein crosslink repair.Mol. Cell. 2016; 64: 704-719Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). SPRTN also drives auto-proteolysis in trans in the presence of ssDNA and double-stranded DNA (dsDNA) (28Li F. Raczynska J.E. Chen Z. Yu H. Structural insight into DNA-dependent activation of human metalloprotease spartan.Cell Rep. 2019; 26: 3336-3346.e3334Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Crystal structure of the SprT domain revealed a metalloprotease subdomain and Zn2+-binding subdomain, which regulate ssDNA binding and protease activity of SPRTN (28Li F. Raczynska J.E. Chen Z. Yu H. Structural insight into DNA-dependent activation of human metalloprotease spartan.Cell Rep. 2019; 26: 3336-3346.e3334Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). SPRTN depletion sensitized cells to treatment with formaldehyde and etoposide, suggesting a role of SPRTN in the repair of formaldehyde-induced DPCs and TOP2-ccs, respectively (17Stingele J. Bellelli R. Alte F. Hewitt G. Sarek G. Maslen S.L. Tsutakawa S.E. Borg A. Kjaer S. Tainer J.A. Skehel J.M. Groll M. Boulton S.J. Mechanism and regulation of DNA-protein crosslink repair by the DNA-dependent metalloprotease SPRTN.Mol. Cell. 2016; 64: 688-703Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 18Vaz B. Popovic M. Newman J.A. Fielden J. Aitkenhead H. Halder S. Singh A.N. Vendrell I. Fischer R. Torrecilla I. Drobnitzky N. Freire R. Amor D.J. Lockhart P.J. Kessler B.M. et al.Metalloprotease SPRTN/DVC1 orchestrates replication-coupled DNA-protein crosslink repair.Mol. Cell. 2016; 64: 704-719Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 19Lopez-Mosqueda J. Maddi K. Prgomet S. Kalayil S. Marinovic-Terzic I. Terzic J. Dikic I. SPRTN is a mammalian DNA-binding metalloprotease that resolves DNA-protein crosslinks.Elife. 2016; 5e21491Crossref PubMed Scopus (65) Google Scholar). In humans, biallelic mutations in SPRTN lead to Ruijs–Aalfs syndrome (RJALS) characterized by genome instability, segmental progeria, and early-onset hepatocellular carcinoma. RJALS patient cells were defective in SPRTN protease activity, displayed defects in replication fork progression and hypersensitivity to DPC-inducing agents (29Lessel D. Vaz B. Halder S. Lockhart P.J. Marinovic-Terzic I. Lopez-Mosqueda J. Philipp M. Sim J.C. Smith K.R. Oehler J. Cabrera E. Freire R. Pope K. Nahid A. Norris F. et al.Mutations in SPRTN cause early onset hepatocellular carcinoma, genomic instability and progeroid features.Nat. Genet. 2014; 46: 1239-1244Crossref PubMed Scopus (110) Google Scholar). Loss of Sprtn in mice resulted in embryonic lethality, while Sprtn hypomorphic mice recapitulated some of the progeroid phenotypes and developed spontaneous tumorigenesis in the liver with increased accumulation of DPCs in the liver tissue. Mouse embryonic fibroblasts from Sprtn hypomorphic mice displayed accumulation of unrepaired TOP1-ccs and were hypersensitive to treatment with DPC-inducing agents (30Maskey R.S. Kim M.S. Baker D.J. Childs B. Malureanu L.A. Jeganathan K.B. Machida Y. van Deursen J.M. Machida Y.J. Spartan deficiency causes genomic instability and progeroid phenotypes.Nat. Commun. 2014; 5: 5744Crossref PubMed Scopus (59) Google Scholar, 31Maskey R.S. Flatten K.S. Sieben C.J. Peterson K.L. Baker D.J. Nam H.J. Kim M.S. Smyrk T.C. Kojima Y. Machida Y. Santiago A. van Deursen J.M. Kaufmann S.H. Machida Y.J. Spartan deficiency causes accumulation of Topoisomerase 1 cleavage complexes and tumorigenesis.Nucleic Acids Res. 2017; 45: 4564-4576Crossref PubMed Scopus (48) Google Scholar). These studies showed that SPRTN metalloprotease repairs replication-coupled DPCs in the genome, thereby protecting cells from DPC-induced genome instability, cancer, and aging. A recent study performed in Xenopus egg extracts showed that both SPRTN and the proteasome can repair replication-coupled DPCs but are activated by distinct mechanisms. The recruitment of the proteasome to DPCs required DPC polyubiquitination, while SPRTN was able to degrade nonubiquitinated DPCs. SPRTN-mediated DPC proteolysis depended on the extension of the nascent DNA strand to within a few nucleotides of the DPC lesion, indicating that polymerase stalling at a DPC on either the leading or lagging strand activates SPRTN. SPRTN depletion impaired TLS following DPC proteolysis in both proteasome-mediated and SPRTN-mediated replication-coupled DPC repair, suggesting that in addition to DPC proteolysis, SPRTN regulates bypass of peptide–DNA adducts by TLS during DNA replication (32Larsen N.B. Gao A.O. Sparks J.L. Gallina I. Wu R.A. Mann M. Raschle M. Walter J.C. Duxin J.P. Replication-coupled DNA-protein crosslink repair by SPRTN and the proteasome in Xenopus egg extracts.Mol. Cell. 2019; 73: 574-588.e577Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). SPRTN is a sequence-nonspecific protease that predominantly cleaves substrates in unstructured regions in the vicinity of lysine, arginine, and serine residues (17Stingele J. Bellelli R. Alte F. Hewitt G. Sarek G. Maslen S.L. Tsutakawa S.E. Borg A. Kjaer S. Tainer J.A. Skehel J.M. Groll M. Boulton S.J. Mechanism and regulation of DNA-protein crosslink repair by the DNA-dependent metalloprotease SPRTN.Mol. Cell. 2016; 64: 688-703Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 18Vaz B. Popovic M. Newman J.A. Fielden J. Aitkenhead H. Halder S. Singh A.N. Vendrell I. Fischer R. Torrecilla I. Drobnitzky N. Freire R. Amor D.J. Lockhart P.J. Kessler B.M. et al.Metalloprotease SPRTN/DVC1 orchestrates replication-coupled DNA-protein crosslink repair.Mol. Cell. 2016; 64: 704-719Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Several mechanisms regulate SPRTN function in DPC repair. SPRTN protease activity is stimulated by DNA binding, while posttranslational modification of SPRTN governs both its protease activity and recruitment to the DPC on chromatin. CHK1 kinase phosphorylates SPRTN at the C-terminal (S373, S374, and S383) and enhances SPRTN protease activity and recruitment to chromatin (33Halder S. Torrecilla I. Burkhalter M.D. Popovic M. Fielden J. Vaz B. Oehler J. Pilger D. Lessel D. Wiseman K. Singh A.N. Vendrell I. Fischer R. Philipp M. Ramadan K. SPRTN protease and checkpoint kinase 1 cross-activation loop safeguards DNA replication.Nat. Commun. 2019; 10: 3142Crossref PubMed Scopus (15) Google Scholar). SPRTN is also monoubiquitinated, which prevents SPRTN access to chromatin and regulates SPRTN protein levels (17Stingele J. Bellelli R. Alte F. Hewitt G. Sarek G. Maslen S.L. Tsutakawa S.E. Borg A. Kjaer S. Tainer J.A. Skehel J.M. Groll M. Boulton S.J. Mechanism and regulation of DNA-protein crosslink repair by the DNA-dependent metalloprotease SPRTN.Mol. Cell. 2016; 64: 688-703Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 23Mosbech A. Gibbs-Seymour I. Kagias K. Thorslund T. Beli P. Povlsen L. Nielsen S.V. Smedegaard S. Sedgwick G. Lukas C. Hartmann-Petersen R. Lukas J. Choudhary C. Pocock R. Bekker-Jensen S. et al.DVC1 (C1orf124) is a DNA damage-targeting p97 adaptor that promotes ubiquitin-dependent responses to replication blocks.Nat. Struct. Mol. Biol. 2012; 19: 1084-1092Crossref PubMed Scopus (123) Google Scholar, 34Zhao S. Kieser A. Li H.Y. Reinking H.K. Weickert P. Euteneuer S. Yaneva D. Acampora A.C. Gotz M.J. Feederle R. Stingele J. A ubiquitin switch controls autocatalytic inactivation of the DNA-protein crosslink repair protease SPRTN.Nucleic Acids Res. 2021; 49: 902-915Crossref PubMed Scopus (4) Google Scholar, 35Huang J. Zhou Q. Gao M. Nowsheen S. Zhao F. Kim W. Zhu Q. Kojima Y. Yin P. Zhang Y. Guo G. Tu X. Deng M. Luo K. Qin B. et al.Tandem deubiquitination and acetylation of SPRTN promotes DNA-protein crosslink repair and protects against aging.Mol. Cell. 2020; 79: 824-835.e825Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Upon DPC induction, ATM/ATR kinase activates VCPIP1/VCIP135 deubiquitinase, which in turn deubiquitinates SPRTN, regulating its chromatin localization. Deubiquitination of SPRTN is a prerequisite for its subsequent acetylation, which promotes SPRTN relocation to chromatin (35Huang J. Zhou Q. Gao M. Nowsheen S. Zhao F. Kim W. Zhu Q. Kojima Y. Yin P. Zhang Y. Guo G. Tu X. Deng M. Luo K. Qin B. et al.Tandem deubiquitination and acetylation of SPRTN promotes DNA-protein crosslink repair and protects against aging.Mol. Cell. 2020; 79: 824-835.e825Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). In contrast, a recent study showed that monoubiquitination of SPRTN does not regulate SPRTN access to chromatin, but instead, promotes SPRTN auto-proteolysis in trans while also priming SPRTN for proteasomal degradation in cis. USP7 deubiquitinase deubiquitinates SPRTN upon DPC induction, antagonizing the autocatalytic cleavage and subsequent inactivation of SPRTN (34Zhao S. Kieser A. Li H.Y. Reinking H.K. Weickert P. Euteneuer S. Yaneva D. Acampora A.C. Gotz M.J. Feederle R. Stingele J. A ubiquitin switch controls autocatalytic inactivation of the DNA-protein crosslink repair protease SPRTN.Nucleic Acids Res. 2021; 49: 902-915Crossref PubMed Scopus (4) Google Scholar). Here, we have identified USP11 as a ubiquitin protease that regulates SPRTN monoubiquitination and auto-proteolysis upon DPC induction. USP11 (ubiquitin carboxyl-terminal hydrolase or ubiquitin-specific protease 11) belongs to the ubiquitin-specific protease (USP or UBP) family of deubiquitinases (DUBs). USP11 participates in processes such as TGFβ signaling, proinflammatory signaling, viral replication, and NF-κB signaling by regulating the protein stability of various targets such as ARID1A, TβRII, CDKN2A, RAE1, XIAP, HPV16-E7, and IκBα (36Luo Q. Wu X. Nan Y. Chang W. Zhao P. Zhang Y. Su D. Liu Z. TRIM32/USP11 balances ARID1A stability and the oncogenic/tumor-suppressive status of squamous cell carcinoma.Cell Rep. 2020; 30: 98-111.e115Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 37Deng T. Yan G. Song X. Xie L. Zhou Y. Li J. Hu X. Li Z. Hu J. Zhang Y. Zhang H. Sun Y. Feng P. Wei D. Hu B. et al.Deubiquitylation and stabilization of p21 by USP11 is critical for cell-cycle progression and DNA damage responses.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: 4678-4683Crossref PubMed Scopus (53) Google Scholar, 38Jacko A.M. Nan L. Li S. Tan J. Zhao J. Kass D.J. Zhao Y. De-ubiquitinating enzyme, USP11, promotes transforming growth factor beta-1 signaling through stabilization of transforming growth factor beta receptor II.Cell Death Dis. 2016; 7e2474Crossref PubMed Scopus (19) Google Scholar, 39Lin C.H. Chang H.S. Yu W.C. USP11 stabilizes HPV-16E7 and further modulates the E7 biological activity.J. Biol. Chem. 2008; 283: 15681-15688Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 40Sun W. Tan X. Shi Y. Xu G. Mao R. Gu X. Fan Y. Yu Y. Burlingame S. Zhang H. Rednam S.P. Lu X. Zhang T. Fu S. Cao G. et al.USP11 negatively regulates TNFalpha-induced NF-kappaB activation by targeting on IkappaBalpha.Cell Signal. 2010; 22: 386-394Crossref PubMed Scopus (69) Google Scholar, 41Stockum A. Snijders A.P. Maertens G.N. USP11 deubiquitinates RAE1 and plays a key role in bipolar spindle formation.PLoS One. 2018; 13e0190513Crossref PubMed Scopus (8) Google Scholar). USP11 also functions in DSB repair, wherein USP11 deubiquitinates H2AX to regulate the recruitment of RAD51 and 53BP1 to damage foci (42Ting X. Xia L. Yang J. He L. Si W. Shang Y. Sun L. USP11 acts as a histone deubiquitinase functioning in chromatin reorganization during DNA repair.Nucleic Acids Res. 2019; 47: 9721-9740Crossref PubMed Scopus (15) Google Scholar, 43Yu M. Liu K. Mao Z. Luo J. Gu W. Zhao W. USP11 is a negative regulator to gammaH2AX ubiquitylation by RNF8/RNF168.J. Biol. Chem. 2016; 291: 959-967Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). USP11 deubiquitinates PALB2 and promotes BRCA1-PALB2-BRCA2 complex formation (44Orthwein A. Noordermeer S.M. Wilson M.D. Landry S. Enchev R.I. Sherker A. Munro M. Pinder J. Salsman J. Dellaire G. Xia B. Peter M. Durocher D. A mechanism for the suppression of homologous recombination in G1 cells.Nature. 2015; 528: 422-426Crossref PubMed Scopus (269) Google Scholar). USP11 confers cellular resistance to PARP1 inhibitors that trap PARP1 to DNA (45Wiltshire T.D. Lovejoy C.A. Wang T. Xia F. O'Connor M.J. Cortez D. Sensitivity to poly(ADP-ribose) polymerase (PARP) inhibition identifies ubiquitin-specific peptidase 11 (USP11) as a regulator of DNA double-strand break repair.J. Biol. Chem. 2010; 285: 14565-14571Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). However, the function of USP11 in DPC repair is not reported. In this study, we show that USP11 is a novel interactor of SPRTN and deubiquitinates SPRTN in cells and in vitro. Depletion of USP11 leads to increased SPRTN auto-proteolysis, an accumulation of unrepaired DPCs, and sensitizes cells to DPC-inducing agents. USP11 cleaves the monoubiquitin on SPRTN upon DPC induction and regulates SPRTN-mediated DPC repair. Monoubiquitinated SPRTN is deubiquitinated upon DNA damage by DPC-inducing agents (17Stingele J. Bellelli R. Alte F. Hewitt G. Sarek G. Maslen S.L. Tsutakawa S.E. Borg A. Kjaer S. Tainer J.A. Skehel J.M. Groll M. Boulton S.J. Mechanism and regulation of DNA-protein crosslink repair by the DNA-dependent metalloprotease SPRTN.Mol. Cell. 2016; 64: 688-703Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Therefore, ubiquitination and deubiquitination of SPRTN are critical for the recruitment of SPRTN to DPC lesions. To identify SPRTN modifiers, we performed tandem-affinity purification and mass spectrometry (TAP-MS) analysis of SFB (S-FLAG-streptavidin binding peptide)-tagged SPRTN expressed in HEK 293T cells. Our MS analysis revealed several known SPRTN interactors, namely PCNA, POLD3, and VCP (Fig. 1A and Table S1). We screened the MS list for proteins that are known to function as a deubiquitinase and identified only one potential SPRTN deubiquitinase, USP11. Similar to POLD3, only one peptide of USP11 was immunoprecipitated in SPRTN MS analysis (Table S2). A reciprocal TAP-MS analysis of SFB-USP11 expressed in HEK 293T cells immunoprecipitated SPRTN in addition to USP7, which is a known USP11 interactor (Fig. 1B and Table S3). We next confirmed SPRTN-USP11 interaction by immunoprecipitation in HEK 293T cells expressing either SFB-USP7, SAMHD1, or SPRTN. USP7 is a known interactor of USP11, while SAMHD1 and SPRTN were identified in SFB-USP11 TAP-MS analysis (Fig. 1B). Endogenous USP11 was immunoprecipitated with SFB-USP7 and SPRTN, but not with SAMHD1 (

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