Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

USP7 is a SUMO deubiquitinase essential for DNA replication

Abstract

Post-translational modification of proteins by ubiquitin (Ub) and Ub-like modifiers regulates DNA replication. We have previously shown that chromatin around replisomes is rich in SUMO and poor in Ub, whereas mature chromatin exhibits an opposite pattern. How this SUMO-rich, Ub-poor environment is maintained at sites of DNA replication in mammalian cells remains unexplored. Here we identify USP7 as a replisome-enriched SUMO deubiquitinase that is essential for DNA replication. By acting on SUMO and SUMOylated proteins, USP7 counteracts their ubiquitination. Inhibition or genetic deletion of USP7 leads to the accumulation of Ub on SUMOylated proteins, which are displaced away from replisomes. Our findings provide a model explaining the differential accumulation of SUMO and Ub at replication forks and identify an essential role of USP7 in DNA replication that should be considered in the development of USP7 inhibitors as anticancer agents.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: USP7 is enriched around replisomes and is essential for DNA replication.
Figure 2: USP7 inhibition reduces replication-fork speed and origin firing.
Figure 3: Identification of SUMO2 as a USP7 target.
Figure 4: USP7 deubiquitinates SUMO2 in vitro and in vivo.
Figure 5: USP7 inhibition leads to the loss of SUMOylated proteins from replisomes.
Figure 6: USP7 deletion mimics the effects of USP7 inhibitors.
Figure 7: A role of USP7 at sites of DNA replication.

References

  1. Lehmann, A.R. Ubiquitin-family modifications in the replication of DNA damage. FEBS Lett. 585, 2772–2779 (2011).

    CAS  PubMed  Google Scholar 

  2. Zhang, W., Qin, Z., Zhang, X. & Xiao, W. Roles of sequential ubiquitination of PCNA in DNA-damage tolerance. FEBS Lett. 585, 2786–2794 (2011).

    CAS  PubMed  Google Scholar 

  3. Havens, C.G. & Walter, J.C. Mechanism of CRL4(Cdt2), a PCNA-dependent E3 ubiquitin ligase. Genes Dev. 25, 1568–1582 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Huang, T.T. et al. Regulation of monoubiquitinated PCNA by DUB autocleavage. Nat. Cell Biol. 8, 339–347 (2006).

    CAS  PubMed  Google Scholar 

  5. Cohn, M.A. et al. A UAF1-containing multisubunit protein complex regulates the Fanconi anemia pathway. Mol. Cell 28, 786–797 (2007).

    CAS  PubMed  Google Scholar 

  6. Nijman, S.M. et al. The deubiquitinating enzyme USP1 regulates the Fanconi anemia pathway. Mol. Cell 17, 331–339 (2005).

    CAS  PubMed  Google Scholar 

  7. Sriramachandran, A.M. & Dohmen, R.J. SUMO-targeted ubiquitin ligases. Biochim. Biophys. Acta 1843, 75–85 (2014).

    CAS  PubMed  Google Scholar 

  8. Galanty, Y., Belotserkovskaya, R., Coates, J. & Jackson, S.P. RNF4, a SUMO-targeted ubiquitin E3 ligase, promotes DNA double-strand break repair. Genes Dev. 26, 1179–1195 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Ragland, R.L. et al. RNF4 and PLK1 are required for replication fork collapse in ATR-deficient cells. Genes Dev. 27, 2259–2273 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Yin, Y. et al. SUMO-targeted ubiquitin E3 ligase RNF4 is required for the response of human cells to DNA damage. Genes Dev. 26, 1196–1208 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Ulrich, H.D. Two-way communications between ubiquitin-like modifiers and DNA. Nat. Struct. Mol. Biol. 21, 317–324 (2014).

    CAS  PubMed  Google Scholar 

  12. Davis, E.J. et al. DVC1 (C1orf124) recruits the p97 protein segregase to sites of DNA damage. Nat. Struct. Mol. Biol. 19, 1093–1100 (2012).

    CAS  PubMed  Google Scholar 

  13. Mosbech, A. et al. DVC1 (C1orf124) is a DNA damage–targeting p97 adaptor that promotes ubiquitin-dependent responses to replication blocks. Nat. Struct. Mol. Biol. 19, 1084–1092 (2012).

    CAS  PubMed  Google Scholar 

  14. Gibbs-Seymour, I. et al. Ubiquitin-SUMO circuitry controls activated fanconi anemia ID complex dosage in response to DNA damage. Mol. Cell 57, 150–164 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Hendriks, I.A., Schimmel, J., Eifler, K., Olsen, J.V. & Vertegaal, A.C. Ubiquitin-specific protease 11 (USP11) deubiquitinates hybrid small ubiquitin-like modifier (SUMO)-ubiquitin chains to counteract RING finger protein 4 (RNF4). J. Biol. Chem. 290, 15526–15537 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Psakhye, I. & Jentsch, S. Protein group modification and synergy in the SUMO pathway as exemplified in DNA repair. Cell 151, 807–820 (2012).

    CAS  PubMed  Google Scholar 

  17. Bursomanno, S. et al. Proteome-wide analysis of SUMO2 targets in response to pathological DNA replication stress in human cells. DNA Repair (Amst.) 25, 84–96 (2015).

    CAS  Google Scholar 

  18. Bursomanno, S., McGouran, J.F., Kessler, B.M., Hickson, I.D. & Liu, Y. Regulation of SUMO2 target proteins by the proteasome in human cells exposed to replication stress. J. Proteome Res. 14, 1687–1699 (2015).

    CAS  PubMed  Google Scholar 

  19. Schimmel, J. et al. Uncovering SUMOylation dynamics during cell-cycle progression reveals FoxM1 as a key mitotic SUMO target protein. Mol. Cell 53, 1053–1066 (2014).

    CAS  PubMed  Google Scholar 

  20. Tammsalu, T. et al. Proteome-wide identification of SUMO2 modification sites. Sci. Signal. 7, rs2 (2014).

    PubMed  PubMed Central  Google Scholar 

  21. Xiao, Z. et al. System-wide analysis of SUMOylation dynamics in response to replication stress reveals novel small ubiquitin-like modified target proteins and acceptor lysines relevant for genome stability. Mol. Cell. Proteomics 14, 1419–1434 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Sirbu, B.M. et al. Analysis of protein dynamics at active, stalled, and collapsed replication forks. Genes Dev. 25, 1320–1327 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Lopez-Contreras, A.J. et al. A proteomic characterization of factors enriched at nascent DNA molecules. Cell Rep. 3, 1105–1116 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Dungrawala, H. et al. The replication checkpoint prevents two types of fork collapse without regulating replisome stability. Mol. Cell 59, 998–1010 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Li, M., Brooks, C.L., Kon, N. & Gu, W. A dynamic role of HAUSP in the p53-Mdm2 pathway. Mol. Cell 13, 879–886 (2004).

    CAS  PubMed  Google Scholar 

  26. Li, M. et al. Deubiquitination of p53 by HAUSP is an important pathway for p53 stabilization. Nature 416, 648–653 (2002).

    CAS  PubMed  Google Scholar 

  27. Altun, M. et al. Activity-based chemical proteomics accelerates inhibitor development for deubiquitylating enzymes. Chem. Biol. 18, 1401–1412 (2011).

    CAS  PubMed  Google Scholar 

  28. Chauhan, D. et al. A small molecule inhibitor of ubiquitin-specific protease-7 induces apoptosis in multiple myeloma cells and overcomes bortezomib resistance. Cancer Cell 22, 345–358 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Reverdy, C. et al. Discovery of specific inhibitors of human USP7/HAUSP deubiquitinating enzyme. Chem. Biol. 19, 467–477 (2012).

    CAS  PubMed  Google Scholar 

  30. Kon, N. et al. Inactivation of HAUSP in vivo modulates p53 function. Oncogene 29, 1270–1279 (2010).

    CAS  PubMed  Google Scholar 

  31. Alonso-de Vega, I., Martín, Y. & Smits, V.A. USP7 controls Chk1 protein stability by direct deubiquitination. Cell Cycle 13, 3921–3926 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Colleran, A. et al. Deubiquitination of NF-κB by ubiquitin-specific protease-7 promotes transcription. Proc. Natl. Acad. Sci. USA 110, 618–623 (2013).

    CAS  PubMed  Google Scholar 

  33. de Bie, P., Zaaroor-Regev, D. & Ciechanover, A. Regulation of the Polycomb protein RING1B ubiquitination by USP7. Biochem. Biophys. Res. Commun. 400, 389–395 (2010).

    CAS  PubMed  Google Scholar 

  34. Faustrup, H., Bekker-Jensen, S., Bartek, J., Lukas, J. & Mailand, N. USP7 counteracts SCFβTrCP- but not APCCdh1-mediated proteolysis of Claspin. J. Cell Biol. 184, 13–19 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Khoronenkova, S.V. & Dianov, G.L. USP7S-dependent inactivation of Mule regulates DNA damage signalling and repair. Nucleic Acids Res. 41, 1750–1756 (2013).

    CAS  PubMed  Google Scholar 

  36. Lecona, E., Narendra, V. & Reinberg, D. USP7 cooperates with SCML2 to regulate the activity of PRC1. Mol. Cell. Biol. 35, 1157–1168 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Maertens, G.N., El Messaoudi-Aubert, S., Elderkin, S., Hiom, K. & Peters, G. Ubiquitin-specific proteases 7 and 11 modulate Polycomb regulation of the INK4a tumour suppressor. EMBO J. 29, 2553–2565 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Qian, J. et al. USP7 modulates UV-induced PCNA monoubiquitination by regulating DNA polymerase eta stability. Oncogene 34, 4791–4796 (2015).

    CAS  PubMed  Google Scholar 

  39. Schwertman, P. et al. UV-sensitive syndrome protein UVSSA recruits USP7 to regulate transcription-coupled repair. Nat. Genet. 44, 598–602 (2012).

    CAS  PubMed  Google Scholar 

  40. Song, M.S. et al. The deubiquitinylation and localization of PTEN are regulated by a HAUSP–PML network. Nature 455, 813–817 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. van der Horst, A. et al. FOXO4 transcriptional activity is regulated by monoubiquitination and USP7/HAUSP. Nat. Cell Biol. 8, 1064–1073 (2006).

    CAS  PubMed  Google Scholar 

  42. Zhang, P. et al. ATM-mediated stabilization of ZEB1 promotes DNA damage response and radioresistance through CHK1. Nat. Cell Biol. 16, 864–875 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Jagannathan, M. et al. A role for USP7 in DNA replication. Mol. Cell. Biol. 34, 132–145 (2014).

    PubMed  PubMed Central  Google Scholar 

  44. Du, Z. et al. DNMT1 stability is regulated by proteins coordinating deubiquitination and acetylation-driven ubiquitination. Sci. Signal. 3, ra80 (2010).

    PubMed  PubMed Central  Google Scholar 

  45. Felle, M. et al. The USP7/Dnmt1 complex stimulates the DNA methylation activity of Dnmt1 and regulates the stability of UHRF1. Nucleic Acids Res. 39, 8355–8365 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Qin, W., Leonhardt, H. & Spada, F. Usp7 and Uhrf1 control ubiquitination and stability of the maintenance DNA methyltransferase Dnmt1. J. Cell. Biochem. 112, 439–444 (2011).

    CAS  PubMed  Google Scholar 

  47. Sowa, M.E., Bennett, E.J., Gygi, S.P. & Harper, J.W. Defining the human deubiquitinating enzyme interaction landscape. Cell 138, 389–403 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Ge, X.Q., Jackson, D.A. & Blow, J.J. Dormant origins licensed by excess Mcm2-7 are required for human cells to survive replicative stress. Genes Dev. 21, 3331–3341 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Ibarra, A., Schwob, E. & Méndez, J. Excess MCM proteins protect human cells from replicative stress by licensing backup origins of replication. Proc. Natl. Acad. Sci. USA 105, 8956–8961 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Kim, W. et al. Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol. Cell 44, 325–340 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Liang, Q. et al. A selective USP1–UAF1 inhibitor links deubiquitination to DNA damage responses. Nat. Chem. Biol. 10, 298–304 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Alabert, C. et al. Nascent chromatin capture proteomics determines chromatin dynamics during DNA replication and identifies unknown fork components. Nat. Cell Biol. 16, 281–293 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Tatham, M.H. et al. RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation. Nat. Cell Biol. 10, 538–546 (2008).

    CAS  PubMed  Google Scholar 

  54. Dantuma, N.P. & Hoppe, T. Growing sphere of influence: Cdc48/p97 orchestrates ubiquitin-dependent extraction from chromatin. Trends Cell Biol. 22, 483–491 (2012).

    CAS  PubMed  Google Scholar 

  55. Magnaghi, P. et al. Covalent and allosteric inhibitors of the ATPase VCP/p97 induce cancer cell death. Nat. Chem. Biol. 9, 548–556 (2013).

    CAS  PubMed  Google Scholar 

  56. Uwada, J. et al. The p150 subunit of CAF-1 causes association of SUMO2/3 with the DNA replication foci. Biochem. Biophys. Res. Commun. 391, 407–413 (2010).

    CAS  PubMed  Google Scholar 

  57. Weinstock, J. et al. Selective dual inhibitors of the cancer-related deubiquitylating proteases USP7 and USP47. ACS Med. Chem. Lett. 3, 789–792 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Toledo, L.I. et al. A cell-based screen identifies ATR inhibitors with synthetic lethal properties for cancer-associated mutations. Nat. Struct. Mol. Biol. 18, 721–727 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Lecona, E. et al. Upregulation of annexin A1 expression by butyrate in human colon adenocarcinoma cells: role of p53, NF-Y, and p38 mitogen-activated protein kinase. Mol. Cell. Biol. 28, 4665–4674 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Méndez, J. & Stillman, B. Chromatin association of human origin recognition complex, cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of prereplication complexes in late mitosis. Mol. Cell. Biol. 20, 8602–8612 (2000).

    PubMed  PubMed Central  Google Scholar 

  61. Ekholm-Reed, S. et al. Deregulation of cyclin E in human cells interferes with prereplication complex assembly. J. Cell Biol. 165, 789–800 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Lecona, E. et al. Polycomb protein SCML2 regulates the cell cycle by binding and modulating CDK/CYCLIN/p21 complexes. PLoS Biol. 11, e1001737 (2013).

    PubMed  PubMed Central  Google Scholar 

  63. López-Contreras, A.J., Gutierrez-Martinez, P., Specks, J., Rodrigo-Perez, S. & Fernandez-Capetillo, O. An extra allele of Chk1 limits oncogene-induced replicative stress and promotes transformation. J. Exp. Med. 209, 455–461 (2012).

    PubMed  PubMed Central  Google Scholar 

  64. Gao, Z. et al. PCGF homologs, CBX proteins, and RYBP define functionally distinct PRC1 family complexes. Mol. Cell 45, 344–356 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Mourón, S. et al. Repriming of DNA synthesis at stalled replication forks by human PrimPol. Nat. Struct. Mol. Biol. 20, 1383–1389 (2013).

    PubMed  Google Scholar 

  66. Jackson, D.A. & Pombo, A. Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells. J. Cell Biol. 140, 1285–1295 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Petermann, E., Orta, M.L., Issaeva, N., Schultz, N. & Helleday, T. Hydroxyurea-stalled replication forks become progressively inactivated and require two different RAD51-mediated pathways for restart and repair. Mol. Cell 37, 492–502 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Wis´niewski, J.R., Zougman, A., Nagaraj, N. & Mann, M. Universal sample preparation method for proteome analysis. Nat. Methods 6, 359–362 (2009).

    Google Scholar 

  69. Wis´niewski, J.R., Zougman, A. & Mann, M. Combination of FASP and StageTip-based fractionation allows in-depth analysis of the hippocampal membrane proteome. J. Proteome Res. 8, 5674–5678 (2009).

    Google Scholar 

  70. Rappsilber, J., Ishihama, Y. & Mann, M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75, 663–670 (2003).

    CAS  PubMed  Google Scholar 

  71. Udeshi, N.D., Mertins, P., Svinkina, T. & Carr, S.A. Large-scale identification of ubiquitination sites by mass spectrometry. Nat. Protoc. 8, 1950–1960 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

Research was funded by Fundación Botín, Banco Santander, through its Santander Universities Global Division, and by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) (SAF2011-23753 and SAF2014-57791-REDC), Worldwide Cancer Research (12-0229), Fundació La Marato de TV3, the Howard Hughes Medical Institute and the European Research Council (ERC-617840) to O.F.-C.; by a Marie-Curie International Outgoing Fellowship (project no. 235705) from the Seventh Framework Programme for Research and Technological Development, Marie Curie Actions, and a grant from MINECO (BFU2014-55168-JIN) that was cofunded by European Regional Development Funds (FEDER) to E.L.; by grants from the Danish Council for Independent Research and the Danish National Research Foundation to A.J.L.-C.; and by a PhD fellowship from MINECO (BES-2012-05 2030) to J.S. We would like to thank W. Gu (Columbia University) for providing Usp7lox/lox MEFs, J. Chen (MD Anderson Cancer Center) for providing Rad18-deficient HCT116 cells and J. Alegre-Cebollada (Spanish National Center for Cardiovascular Research) for help in the use of PyMOL software.

Author information

Authors and Affiliations

Authors

Contributions

E.L. participated in most of the experiments of this study and wrote the paper. S.R.-A. and J. Mendez performed DNA fiber analyses. I.R. and J. Muñoz assisted with proteomics. J.S. and M.M. assisted with IF and HTM analyses. A.J.L.-C. assisted with iPOND experiments. O.F.-C. coordinated the study and wrote the paper.

Corresponding authors

Correspondence to Emilio Lecona or Oscar Fernandez-Capetillo.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 USP7 inhibition stops DNA replication in a time- and dose-dependent manner.

(a-b) HCT116 cells were treated with increasing concentrations of P22077 or DMSO as a control for the indicated times. After incubation with 20 μM EdU for 30 min, cells were fixed and analyzed by HTM that quantified EdU (a) and γH2AX (b) levels per individual nucleus. DAPI was used to identify nuclei.

(c) Whole cell extracts of HCT116 cells treated with increasing concentrations of P22077 or DMSO (C) for the indicated times were analyzed by WB with antibodies against USP7, p21, p53 and MCM4.

Supplementary Figure 2 The effects of USP7 inhibition on DNA replication and replicative stress are independent of p53.

(a-b) Wild type (WT) and p53-deficient (p53 KO) MEF were treated with increasing concentrations of P22077 or DMSO (C) for the indicated times. After incubation with 20 μM EdU for 30 min, cells were fixed and analyzed by HTM that quantified EdU (a) and γH2AX (b) levels per individual nucleus.

(c) Representative IF examples illustrating the levels of EdU (green) and γH2AX (red) observed in WT and p53 KO MEF after 4 h of treatment. DAPI (blue) was used to identify nuclei. Scale bar, 20 μm.

Supplementary Figure 3 Two independent USP7 inhibitors recapitulate the effects of P22077, independently of PCNA ubiquitination.

(a-b) HCT116 cells were treated with increasing concentrations of P5091 (a) or HBX19818 (b), using DMSO as a control for the indicated times. After incubation with 20 μM EdU for 30 min, cells were fixed and analyzed by HTM that quantified EdU levels per individual nucleus. Equivalent results were obtained in two independent experiments.

(c-d) Whole cell extracts of HCT116 cells treated with increasing concentrations of P5091 or DMSO as a control for the indicated times were analyzed by WB with antibodies against USP7, PCNA, total and phosphorylated Chk1 (S345), and total and phosphorylated RPA2 (S4/S8) (c), or with antibodies against p53, p21 and MCM7 (d). Equivalent results were obtained in two independent experiments.

(e) Whole cell extracts of HCT116 wild-type (WT) and Rad18 KO cells treated with increasing concentrations of P22077 or DMSO (C) for the indicated times were analyzed by WB with antibodies against USP7, PCNA, total and phosphorylated Chk1 (S345), and total and phosphorylated RPA2 (S4/S8), and p21. Equivalent results were obtained in two independent experiments.

Supplementary Figure 4 Effect of USP7 overexpression on cell-cycle progression and origin firing.

(a) Cell cycle distribution of 293T-REx cells OneStrep-FLAG-HA-USP7 after being incubated for 1 day with (+Dox) or without (-Dox) 1 μg/ml doxycycline, measured by FACS staining with propidium iodide (PI) and BrdU. The percentage of BrdU cells is shown in (b).

(c) DNA fibers were extracted from 293T-REx OneStrep-FLAG-HA-USP7 cells incubated for 1 day with (+Dox) or without (-Dox) 1 μg/ml doxycycline and then sequentially treated with CldU and IdU. The firing of new origins was quantified as in Fig. 2e. ** p<0.01, t-test, two tails.

Supplementary Figure 5 Mass spectrometry analysis of the effect of USP7 on poly-SUMO2 and polyubiquitin chains.

(a) The intensity of the peptides for ubiquitin (black) and SUMO2 (grey) in the Ub-SUMO2X3 and the SUMO2X3 bands is shown.

(b) Di-Gly containing peptides for SUMO2 were identified and the cumulative intensity of all these peptides is shown for both bands.

(c) Poly-Ubiquitin (3-7) K48 or K63 chains were incubated with 25-100 nM USP7 or 100 nM USP1/UAF1 or in the absence of enzyme (C) for 2h at 37 °C. The products of the reaction were detected by Coomassie staining. The position of the different chains of Ubiquitin is indicated, together with the position of USP7 and the USP1/UAF1 dimmer.

(d) Poly-SUMO2 (3-8) chains were incubated with 20 nM USP7 or in the absence of enzyme (C) and the levels of SUMO2 were measured by WB.

Supplementary Figure 6 Replication stress does not induce accumulation of SUMOylated proteins on chromatin.

(a) Representative IF images of U2OS cells treated with 50 μM P22077 or DMSO (C) for 4 h. Chromatin-bound levels of SUMO2 (red) were analyzed by a IF protocol that previously extracts the nuclear-soluble fraction of proteins. Besides this isolated example, the overall levels of chromatin-bound SUMO2/3 per individual nucleus as quantified by HTM are shown in Fig. 4e.

(b) HCT116 cells were treated with DMSO (C), 50 μM P22077 (P22) or 2-5 mM hydroxyurea (HU) for 2 h (left) or 4 h (right). The chromatin fraction was extracted and analyzed by Western blot with antibodies against SUMO2/3 or ubiquitin.

Supplementary Figure 7 Effects of inhibition of USP7 and/or p97 on DNA replication and effects of AdCre infection on WT MEFs.

(a-b) HCT116 cells were treated with 50 μM P22077 (P22), 5 μM NMS873 (NMS), both (P22-NMS) or DMSO as a control (C), for the indicated times. Chromatin fractions were analyzed by WB with antibodies against SUMO2/3 and USP7 (a), or Ub and histone H2A (b). Equivalent results were obtained in two independent experiments.

(c) HCT116 cells treated as in (a) were incubated with 20 μM EdU for 30 min. After incubation with 20 μM EdU for 30 min, cells were fixed and analyzed by HTM that quantified EdU levels per individual nucleus. The experiment was repeated three times and one representative experiment is shown.

(d) Western blot analysis of whole cell extracts from WT MEF mock infected (C) or infected with AdCre for 4 days. The levels of USP7 and CDK2 were measured using specific antibodies.

(e-f) DNA fibers were extracted 4 days after mock or AdCre infection of WT MEF and the fork rate (e) and percentage of origin firing (f) were measured. The experiments were repeated three times; the pool of the three experiments (fork rate) or the average (origin firing) is shown.

(g) Chromatin fraction of mock or AdCre infected WT MEF were assayed by Western blot with antibodies against SUMO2/3. Equivalent results were obtained in two independent experiments

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 to 1–7 (PDF 1088 kb)

Supplementary Data Set 1

Uncropped gel images (PDF 2760 kb)

Supplementary Table 1

iPOND data (XLS 370 kb)

Supplementary Table 2

Quantitative K-ɛ-GG site analysis for two HCT human cell biological replicates (XLSX 2390 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Lecona, E., Rodriguez-Acebes, S., Specks, J. et al. USP7 is a SUMO deubiquitinase essential for DNA replication. Nat Struct Mol Biol 23, 270–277 (2016). https://doi.org/10.1038/nsmb.3185

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.3185

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing