Ubiquitin-directed AAA+ ATPase p97/VCP unfolds stable proteins crosslinked to DNA for proteolysis by SPRTN

Abstract

The protease SPRTN degrades DNA-protein crosslinks (DPCs) that threaten genome stability. SPRTN has been connected to the ubiquitin-directed protein unfoldase p97 (also called VCP or Cdc48), but a functional cooperation has not been demonstrated directly. Here, we biochemically reconstituted p97-assisted proteolysis with purified proteins and showed that p97 targets ubiquitin-modified DPCs and unfolds them to prepare them for proteolysis by SPRTN. We demonstrate that purified SPRTN alone was unable to degrade a tightly-folded Eos fluorescent reporter protein even when Eos was crosslinked to DNA (Eos-DPC). However, when present, p97 unfolded poly-ubiquitinated Eos-DPC in a manner requiring its ubiquitin adapter, Ufd1-Npl4. Notably, we show that, in cooperation with p97 and Ufd1-Npl4, SPRTN proteolyzed unfolded Eos-DPC, which relied on recognition of the DNA-crosslink by SPRTN. In a simplified unfolding assay, we further demonstrate that p97, while unfolding a protein substrate, can surmount the obstacle of a DNA crosslink site in the substrate. Thus, our data demonstrate that p97, in conjunction with Ufd1-Npl4, assists SPRTN-mediated proteolysis of tightly-folded proteins crosslinked to DNA, even threading bulky protein-DNA adducts. These findings will be relevant for understanding how cells handle DPCs to ensure genome stability and for designing strategies that target p97 in combination cancer therapy. The protease SPRTN degrades DNA-protein crosslinks (DPCs) that threaten genome stability. SPRTN has been connected to the ubiquitin-directed protein unfoldase p97 (also called VCP or Cdc48), but a functional cooperation has not been demonstrated directly. Here, we biochemically reconstituted p97-assisted proteolysis with purified proteins and showed that p97 targets ubiquitin-modified DPCs and unfolds them to prepare them for proteolysis by SPRTN. We demonstrate that purified SPRTN alone was unable to degrade a tightly-folded Eos fluorescent reporter protein even when Eos was crosslinked to DNA (Eos-DPC). However, when present, p97 unfolded poly-ubiquitinated Eos-DPC in a manner requiring its ubiquitin adapter, Ufd1-Npl4. Notably, we show that, in cooperation with p97 and Ufd1-Npl4, SPRTN proteolyzed unfolded Eos-DPC, which relied on recognition of the DNA-crosslink by SPRTN. In a simplified unfolding assay, we further demonstrate that p97, while unfolding a protein substrate, can surmount the obstacle of a DNA crosslink site in the substrate. Thus, our data demonstrate that p97, in conjunction with Ufd1-Npl4, assists SPRTN-mediated proteolysis of tightly-folded proteins crosslinked to DNA, even threading bulky protein-DNA adducts. These findings will be relevant for understanding how cells handle DPCs to ensure genome stability and for designing strategies that target p97 in combination cancer therapy. Chromatin-associated proteins commonly form covalent crosslinks with the DNA, termed DNA-protein crosslinks (DPCs) that are induced by physical or chemical crosslinkers and can even occur naturally (1Ruggiano A. Ramadan K. DNA-protein crosslink proteases in genome stability.Commun. Biol. 2021; 4: 11Crossref PubMed Scopus (17) Google Scholar, 2Reinking H.K. Hofmann K. Stingele J. Function and evolution of the DNA-protein crosslink proteases Wss1 and SPRTN.DNA Repair (Amst). 2020; 88: 102822Crossref PubMed Scopus (11) Google Scholar). DPCs represent bulky barriers that interfere with transcription, DNA replication and repair, and therefore threaten cell survival and genome stability. DPCs can be removed by dedicated metalloproteases such as SPRTN (SprT-like N-terminal domain, also known as Spartan or DVC1), its yeast counterpart Wss1, or related proteases (1Ruggiano A. Ramadan K. DNA-protein crosslink proteases in genome stability.Commun. Biol. 2021; 4: 11Crossref PubMed Scopus (17) Google Scholar, 2Reinking H.K. Hofmann K. Stingele J. Function and evolution of the DNA-protein crosslink proteases Wss1 and SPRTN.DNA Repair (Amst). 2020; 88: 102822Crossref PubMed Scopus (11) Google Scholar, 3Stingele 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 (169) Google Scholar, 4Stingele 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 (122) Google Scholar, 5Vaz 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 (130) Google Scholar, 6Lopez-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 (83) Google Scholar, 7Larsen 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.e7Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). DPC proteases do not have any defined substrate sequence preference consistent with the diversity of proteins that form DPCs. However, proteolytic activity of SPRTN requires recognition of the crosslinked DNA by a zinc-binding domain (ZBD) and a basic region (BR) in SPRTN to ensure specificity for DPCs (8Li F. Raczynska J.E. Chen Z. Yu H. Structural insight into DNA-dependent activation of human metalloprotease spartan.Cell Rep. 2019; 26: 3336-3346.e4Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 9Reinking H.K. Kang H.S. Gotz M.J. Li H.Y. Kieser A. Zhao S. Acampora A.C. Weickert P. Fessler E. Jae L.T. Sattler M. Stingele J. DNA structure-specific cleavage of DNA-protein crosslinks by the SPRTN protease.Mol. Cell. 2020; 80: 102-113.e6Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Consistent with its central role, SPRTN is essential in mice while mutations in SPRTN cause severe genetic disorders in humans (10Lessel 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 (126) Google Scholar, 11Maskey 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 (68) Google Scholar). In vitro assays that directly monitor proteolysis of DPCs by SPRTN have relied on structurally unstable reporter proteins (9Reinking H.K. Kang H.S. Gotz M.J. Li H.Y. Kieser A. Zhao S. Acampora A.C. Weickert P. Fessler E. Jae L.T. Sattler M. Stingele J. DNA structure-specific cleavage of DNA-protein crosslinks by the SPRTN protease.Mol. Cell. 2020; 80: 102-113.e6Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar), raising the question if and how tightly folded proteins crosslinked to DNA are proteolyzed by SPRTN. SPRTN and Wss1 have been connected in cells to the AAA+ ATPase p97 (also called VCP or Cdc48 in yeast) (3Stingele 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 (169) Google Scholar, 12Balakirev M.Y. Mullally J.E. Favier A. Assard N. Sulpice E. Lindsey D.F. Rulina A.V. Gidrol X. Wilkinson K.D. Wss1 metalloprotease partners with Cdc48/Doa1 in processing genotoxic SUMO conjugates.Elife. 2015; 4e06763Crossref PubMed Scopus (55) Google Scholar, 13Mosbech 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 (131) Google Scholar, 14Davis 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 (111) Google Scholar). Of note, p97 is a protein unfolding machine and therefore may assist SPRTN by unfolding tightly folded DPCs for proteolysis. The hexameric p97 is best known for unfolding ubiquitylated proteins and preparing them for proteolysis in the proteasome (15van den Boom J. Meyer H. VCP/p97-Mediated unfolding as a principle in protein homeostasis and signaling.Mol. Cell. 2018; 69: 182-194Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 16Ye Y. Tang W.K. Zhang T. Xia D. A mighty "protein extractor" of the cell: Structure and function of the p97/CDC48 ATPase.Front. Mol. Biosci. 2017; 4: 39Crossref PubMed Scopus (94) Google Scholar). Notably, p97 unfolds even tightly folded proteins lacking any disordered regions, which are otherwise required for the proteasome in the of p97 C. A. The Cdc48 unfoldase protein for by the Biol. 2019; PubMed Scopus Google Scholar). ubiquitin-directed unfolding by p97, ubiquitin as a unfolding that is by the ubiquitin to p97 Y. G. of ubiquitin by the mammalian p97 adaptor and J. PubMed Scopus Google Scholar, Z. J.A. processing by the Cdc48 ATPase is by ubiquitin 2019; PubMed Scopus Google Scholar). However, not recruits the ubiquitylated substrate to p97 but also of the ubiquitin into the central of the p97 Z. J.A. processing by the Cdc48 ATPase is by ubiquitin 2019; PubMed Scopus Google Scholar). p97 ubiquitin and the substrate its central unfolding the substrate for in the proteasome of substrate processing by the Cdc48 ATPase 2017; Full Text Full Text PDF PubMed Scopus Google Scholar, and unfoldase activity of is by a that causes S. A. 2017; PubMed Scopus Google Scholar). In we unfolding can directly assist also proteolysis of tightly folded DPCs. We that, in unfolding by p97 may be by ubiquitin Ufd1-Npl4. that SPRTN and a of (1Ruggiano A. Ramadan K. DNA-protein crosslink proteases in genome stability.Commun. Biol. 2021; 4: 11Crossref PubMed Scopus (17) Google Scholar, 2Reinking H.K. Hofmann K. Stingele J. Function and evolution of the DNA-protein crosslink proteases Wss1 and SPRTN.DNA Repair (Amst). 2020; 88: 102822Crossref PubMed Scopus (11) Google Scholar), SPRTN in principle as a substrate for with purified we show directly that unfolding DPCs for proteolysis by SPRTN. we that p97 and SPRTN alone do not but the that substrate Thus, p97 proteolysis of DPCs by SPRTN in ubiquitin and and can occur of the of the cooperation p97 and we to a substrate reporter protein that a DNA crosslink required for by SPRTN with for by p97, as as for unfolding and proteolysis. The of unfolding is by of the substrate, and assays also involved in DPC proteolysis 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 (111) Google Scholar). unfolding by p97 can be with a to the fluorescent reporter that is C. A. The Cdc48 unfoldase protein for by the Biol. 2019; PubMed Scopus Google Scholar, of substrate processing by the Cdc48 ATPase 2017; Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. van den Boom J. M. J. I. J. F. M. A. M. Meyer H. by a p97 Cell. 2018; Full Text Full Text PDF PubMed Scopus Google Scholar). of the reporter protein can be directly by the of Eos A in Eos induced by in a to and of of Eos In the Eos of therefore on proteolysis. a we the reporter protein in A and The in of the protein and therefore in in to the in A and by we crosslinked a DNA to in The was with a in a which is by SPRTN and its protease activity (9Reinking H.K. Kang H.S. Gotz M.J. Li H.Y. Kieser A. Zhao S. Acampora A.C. Weickert P. Fessler E. Jae L.T. Sattler M. Stingele J. DNA structure-specific cleavage of DNA-protein crosslinks by the SPRTN protease.Mol. Cell. 2020; 80: 102-113.e6Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). are in the of the site of the the DNA crosslink to the as as to the of to a in we a DNA to the Consistent with the DNA crosslink to we of the crosslinked in of the A and In the we the reporter substrate with ubiquitin to by was with a protein that the site in the reporter and unfoldase activity of is by a that causes S. A. 2017; PubMed Scopus Google Scholar). in a of reporter to a in the that was for the on the DNA as A and the of the but was with the was in the in a form with of SPRTN and p97 have been in but SPRTN can as a substrate ubiquitin for p97 or to with the ubiquitin of p97 is SPRTN and to with p97 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 (68) Google Scholar, 13Mosbech 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 (131) Google raising the question as to SPRTN and can p97 the we a with purified in which we p97 and SPRTN with of Ufd1-Npl4. We SPRTN and p97 not in the of that not with SPRTN for p97 with the but not SPRTN alone that and SPRTN to p97, by with the p97 Thus, is consistent with DPC proteolysis by SPRTN by SPRTN activity can be by the of proteolytic of its DPC substrate (9Reinking H.K. Kang H.S. Gotz M.J. Li H.Y. Kieser A. Zhao S. Acampora A.C. Weickert P. Fessler E. Jae L.T. Sattler M. Stingele J. DNA structure-specific cleavage of DNA-protein crosslinks by the SPRTN protease.Mol. Cell. 2020; 80: 102-113.e6Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). We therefore SPRTN proteolytic of our reporter substrate protein and that was by We our substrate alone or with SPRTN and in the of of to the of unfolding activity by The the of on and to protein and and In the of the of the substrate and the was as in the In the of SPRTN a cleavage was with of is consistent with Eos N-terminal with the ubiquitin was by SPRTN. that the was Eos The that that the tightly folded Eos was to cleavage by SPRTN. Of note, in the of of p97 unfolding a was and The that the of and the DNA crosslink that was a the Eos The was not when SPRTN or p97 was and that proteolysis was by the p97 or and that p97 unfolding activity was required for the of proteolytic Of note, SPRTN-mediated Eos proteolysis was on the p97 ubiquitin and that substrate protein and was required for proteolysis to occur and that SPRTN not as a substrate of and to p97 that p97 unfolding activity was required and that was to SPRTN-mediated proteolysis. further the of substrate we of Eos the We the that our substrate as as Eos with and for of Eos by p97 to a of Eos In Eos unfolding to in by a of unfolding and We for our substrate reporter by the that the unfolded unfolding of a substrate protein by the PubMed Scopus Google a protease is any of can therefore be to proteolysis C. A. The Cdc48 unfoldase protein for by the Biol. 2019; PubMed Scopus Google Scholar). Consistent with that in a the as as p97, Ufd1-Npl4, and we a of in the that was to our reporter protein Of note, of was not by of SPRTN consistent with unfolding for and that p97 unfolding was In for p97 alone a in as was not by a in of SPRTN to the unfolding to a of that the substrate was proteolyzed These demonstrate that, for tightly folded protein proteolysis by SPRTN is to protein unfolding and requires the recognition of the ubiquitin by Ufd1-Npl4. SPRTN specificity for DPCs by of the DNA its and (9Reinking H.K. Kang H.S. Gotz M.J. Li H.Y. Kieser A. Zhao S. Acampora A.C. Weickert P. Fessler E. Jae L.T. Sattler M. Stingele J. DNA structure-specific cleavage of DNA-protein crosslinks by the SPRTN protease.Mol. Cell. 2020; 80: 102-113.e6Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). substrate proteolysis by SPRTN was on DNA recognition by SPRTN we assays with SPRTN lacking the and and to SPRTN. Notably, proteolysis was by the mutations that DNA recognition was essential and that SPRTN its for DPCs even for proteins unfolded by the was also with SPRTN the in the 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 (122) Google which as SPRTN relevant for a that to p97 and a domain that ubiquitin 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 (122) Google Scholar, J. Gao M. S. Zhao F. Kim Y. P. Zhang Y. G. X. M. K. B. et and of SPRTN promotes DNA-protein crosslink repair and Cell. 2020; Full Text Full Text PDF PubMed Scopus Google Scholar). of the domain a Notably, mutations of the but and even the combination with the not further proteolytic activity We the of on SPRTN to p97 by in vitro by that, while is required to the ubiquitin of the substrate, ubiquitin and p97 by SPRTN to of the but is not essential in the in vitro p97 unfolds proteins by threading them the central of the p97 of substrate processing by the Cdc48 ATPase 2017; Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. van den Boom J. M. J. I. J. F. M. A. M. Meyer H. by a p97 Cell. 2018; Full Text Full Text PDF PubMed Scopus Google Scholar). The that p97 can as the question as to how p97 when the DNA crosslink site in a we to a simplified unfolding that not on but a defined site a substrate protein In p97, in with substrate by targets by to a of protein and by M. J. van den Boom J. M. J. I. J. F. M. A. M. Meyer H. by a p97 Cell. 2018; Full Text Full Text PDF PubMed Scopus Google Scholar). threading its central p97 threading is recognition site in and to the reporter to the of den Boom J. B. H. M. D. F. M. A. Meyer H. substrate by Struct. Mol. Biol. 2021; PubMed Scopus Google Scholar). the of DNA we a domain Eos and and the protein as a with and for by A of the purified protein was further by the domain to a DNA which in a of the proteins in We unfolding of with by as The of the domain to DNA the unfolding site in and the reporter that protein processing by p97 to the DNA crosslink unfolding of Eos can be substrate proteins unfolded of to DNA or not that p97 can surmount a DNA crosslink site while processing substrate In we that SPRTN with the p97 unfolding machine to tightly folded proteins crosslinked to DNA that SPRTN alone is unable to degrade In our of the the of to protein unfolding by p97 and with proteolysis we demonstrate that p97-assisted proteolysis by SPRTN on substrate unfolding by p97 and on of the substrate by the ubiquitin that SPRTN alone as a ubiquitin substrate for p97, the that SPRTN a domain as as a that the substrate and can be by the that substrate such as not p97 to the ubiquitylated substrate but also have to the substrate into the central of p97 for unfolding Z. J.A. processing by the Cdc48 ATPase is by ubiquitin 2019; PubMed Scopus Google Scholar, M. van den Boom J. J. A. F. M. Meyer H. by p97 is recognition of and by the p97 Mol. Biol. 2020; PubMed Scopus Google Scholar). Consistent with a of in SPRTN-mediated was to with SPRTN with p97 in cells 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 (111) Google Scholar). The that the ubiquitin is required also that SPRTN DPC to p97 by to the DNA of DPCs. if a tightly folded protein is crosslinked to DNA, can to the and unfolding to assist SPRTN in proteolysis. the protein has been as a p97 substrate in SPRTN-mediated proteolysis of the cleavage J. K. Torrecilla I. Li S. S. A. Singh A. Freire R. S. E. Vendrell I. Fischer R. Kessler B.M. et and SPRTN-mediated of Commun. 2020; PubMed Scopus Google Scholar), which is a form of However, is for the cleavage J. K. Torrecilla I. Li S. S. A. Singh A. Freire R. S. E. Vendrell I. Fischer R. Kessler B.M. et and SPRTN-mediated of Commun. 2020; PubMed Scopus Google and therefore for the diversity of DPCs. We that the that SPRTN to the p97 not to p97-assisted proteolysis by SPRTN. that physical of SPRTN with p97 is for the of protein unfolding by p97 with proteolysis by SPRTN. In the domain in SPRTN is not further a of SPRTN as a ubiquitin is consistent with the that ubiquitin of the domain in SPRTN has been connected to regulation of SPRTN by J. Gao M. S. Zhao F. Kim Y. P. Zhang Y. G. X. M. K. B. et and of SPRTN promotes DNA-protein crosslink repair and Cell. 2020; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Kieser A. Li H.Y. H.K. Weickert P. S. D. Acampora A.C. Gotz M.J. R. Stingele J. A ubiquitin of the DNA-protein crosslink repair protease 2021; PubMed Scopus Google Scholar), which not occur in our simplified The unfolding of DPCs the question as to how DPCs are by Of note, by the DNA crosslink a unfolding site and the unfolding reporter we demonstrate that p97 can surmount the protein crosslink site substrate protein on is that p97 the protein with the DNA the central of the p97 of a is not of p97 can protein its and even ubiquitin Z. J.A. processing by the Cdc48 ATPase is by ubiquitin 2019; PubMed Scopus Google Scholar, den Boom J. B. H. M. D. F. M. A. Meyer H. substrate by Struct. Mol. Biol. 2021; PubMed Scopus Google Scholar). p97 has been to the to bulky while the is in the Z. Li H. D. J.A. J.A. of protein by the hexameric Cdc48 Cell. 2021; Full Text Full Text PDF PubMed Scopus Google Scholar). we demonstrate that proteolysis by SPRTN of the unfolded substrate requires the DNA recognition of SPRTN that SPRTN for DPCs even when with our data the of p97-assisted proteolysis of DPC by SPRTN in show that p97 with proteases the proteasome for protein The of human SPRTN was into A or was to SPRTN by and of SPRTN by was by in and unfoldase activity of is by a that causes S. A. 2017; PubMed Scopus Google with M. H. Zhang Y. Yu J. Wu L. Chen J. B. J. Y. Zhang J. P. T. of and fluorescent 2012; PubMed Scopus Google and In the the R. P. F. C. J.E. in and of 2012; PubMed Scopus Google the site was by a site and by a by In the and unfoldase activity of is by a that causes S. A. 2017; PubMed Scopus Google Scholar), the site was by a site in by DNA for a of and for a of for protein into or cells and in was induced by of and in with cells for with by and for was a and with of with with directly a and with of SPRTN was by the SPRTN was further purified by a in with of the was by a as was a and with with and proteins in and as in or with for and and as for SPRTN of was by was to the form by for on a as M. J. van den Boom J. M. J. I. J. F. M. A. M. Meyer H. by a p97 Cell. 2018; Full Text Full Text PDF PubMed Scopus Google Scholar). ubiquitin was in and in was for of and for The was in ubiquitin and a was was to and ubiquitin was with in and and of and was as M. J. van den Boom J. M. J. I. J. F. M. A. M. Meyer H. by a p97 Cell. 2018; Full Text Full Text PDF PubMed Scopus Google Scholar, den Boom J. B. H. M. D. F. M. A. Meyer H. substrate by Struct. Mol. Biol. 2021; PubMed Scopus Google was in with or with of purified with in crosslink and with for in crosslink crosslink was a of DNA Eos protein was by a in DNA with was with of in was by of and removed by and the was in with was for with of DNA DPC substrate was as and unfoldase activity of is by a that causes S. A. 2017; PubMed Scopus Google Scholar). was with ubiquitin and the substrate was purified ubiquitin by and as p97 unfolding activity and SPRTN proteolytic activity was by in with DPC substrate p97 and in for the was by of SPRTN and was in a to the data of SPRTN and was The proteolytic activity of SPRTN was by DPC substrate was with p97 SPRTN and in for the was by of for and was with p97 or and with or and for in with with proteins by in The for and of a of p97 and as with for in with with and by in data are the and The A. J. M. and A. F. K. J. B. and H. M. A. J. M. A. F. and H. M. A. J. M. A. F. and H. M. data H. M. H. M. A. J. M. A. F. and H. M. and J. B. M. K. H. M. was by the and to H. M.

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