Regulation of monoubiquitinated PCNA by DUB autocleavage

Article metrics

  • An Erratum to this article was published on 01 April 2006


Monoubiquitination is a reversible post-translational protein modification that has an important regulatory function in many biological processes, including DNA repair. Deubiquitinating enzymes (DUBs) are proteases that are negative regulators of monoubiquitination, but little is known about their regulation and contribution to the control of conjugated-substrate levels. Here, we show that the DUB ubiquitin specific protease 1 (USP1) deubiquitinates the DNA replication processivity factor, PCNA, as a safeguard against error-prone translesion synthesis (TLS) of DNA. Ultraviolet (UV) irradiation inactivates USP1 through an autocleavage event, thus enabling monoubiquitinated PCNA to accumulate and to activate TLS. Significantly, the site of USP1 cleavage is immediately after a conserved internal ubiquitin-like diglycine (Gly–Gly) motif. This mechanism is reminiscent of the processing of precursors of ubiquitin and ubiquitin-like modifiers by DUBs. Our results define a regulatory mechanism for protein ubiquitination that involves the signal-induced degradation of an inhibitory DUB.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Knockdown of USP1 increases PCNA monoubiquitination.
Figure 2: UV damage degrades USP1 and increases PCNA monoubiquitination.
Figure 3: Inhibition of diverse DNA repair pathways does not affect UV-induced USP1 degradation.
Figure 4: Degradation of USP1 requires its own catalytic activity.
Figure 5: The conserved diglycine motif of USP1 is required for its autocleavage.
Figure 6: Increased mutation frequency in cells depleted of USP1.


  1. 1

    Hicke, L. Protein regulation by monoubiquitin. Nature Rev. Mol. Cell Biol. 2, 195–201 (2001).

  2. 2

    Pickart, C. M. Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 70, 503–533 (2001).

  3. 3

    Wilkinson, K. D. Ubiquitination and deubiquitination: targeting of proteins for degradation by the proteasome. Semin. Cell Dev. Biol. 11, 141–148 (2000).

  4. 4

    D'Andrea, A. & Pellman, D. Deubiquitinating enzymes: a new class of biological regulators. Crit. Rev. Biochem. Mol. Biol. 33, 337–352 (1998).

  5. 5

    Friedberg, E. C., Lehmann, A. R. & Fuchs, R. P. Trading places: how do DNA polymerases switch during translesion DNA synthesis? Mol. Cell 18, 499–505 (2005).

  6. 6

    Hoege, C. et al. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419, 135–141 (2002).

  7. 7

    Kannouche, P. L., Wing, J. & Lehmann, A. R. Interaction of human DNA polymerase η with monoubiquitinated PCNA: a possible mechanism for the polymerase switch in response to DNA damage. Mol. Cell 14, 491–500 (2004).

  8. 8

    Watanabe, K. et al. Rad18 guides polη to replication stalling sites through physical interaction and PCNA monoubiquitination. EMBO J. 23, 3886–3896 (2004).

  9. 9

    Friedberg, E. C., Wagner, R. & Radman, M. Specialized DNA polymerases, cellular survival, and the genesis of mutations. Science 296, 1627–1630 (2002).

  10. 10

    Kusumoto, R. et al. DNA binding properties of human DNA polymerase η: implications for fidelity and polymerase switching of translesion synthesis. Genes Cells 9, 1139–1150 (2004).

  11. 11

    Kunkel, T. A. DNA replication fidelity. J. Biol. Chem. 279, 16895–16898 (2004).

  12. 12

    Bienko, M. et al. Ubiquitin-binding domains in Y-family polymerases regulate translesion synthesis. Science 310, 1821–1824 (2005).

  13. 13

    Garg, P. & Burgers, P. M. Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases η and REV1. Proc. Natl Acad. Sci. USA 102, 18361–18366 (2005).

  14. 14

    Amerik, A. Y. & Hochstrasser, M. Mechanism and function of deubiquitinating enzymes. Biochim. Biophys. Acta. 1695, 189–207 (2004).

  15. 15

    Nijman, S. M. et al. A genomic and functional inventory of deubiquitinating enzymes. Cell 123, 773–786 (2005).

  16. 16

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

  17. 17

    Howlett, N. G. et al. The Fanconi anemia pathway is required for the DNA replication stress response and for the regulation of common fragile site stability. Hum. Mol. Genet. 14, 693–701 (2005).

  18. 18

    Hussain, S. et al. Direct interaction of FANCD2 with BRCA2 in DNA damage response pathways. Hum. Mol. Genet. 13, 1241–1248 (2004).

  19. 19

    Stelter, P. & Ulrich, H. D. Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation. Nature 425, 188–191 (2003).

  20. 20

    Garcia-Higuera, I. et al. Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway. Mol. Cell 7, 249–262 (2001).

  21. 21

    D'Andrea, A. D. & Grompe, M. The Fanconi anaemia/BRCA pathway. Nature Rev. Cancer 3, 23–34 (2003).

  22. 22

    Kennedy, R. D. & D'Andrea, A. D. The Fanconi Anemia/BRCA pathway: new faces in the crowd. Genes Dev. 19, 2925–2940 (2005).

  23. 23

    Meetei, A. R. et al. A novel ubiquitin ligase is deficient in Fanconi anemia. Nature Genet. 35, 165–170 (2003).

  24. 24

    Borodovsky, A. et al. Chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family. Chem. Biol. 9, 1149–1159 (2002).

  25. 25

    Tagami, H., Ray-Gallet, D., Almouzni, G. & Nakatani, Y. Histone H3.1 and H3.3 complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis. Cell 116, 51–61 (2004).

  26. 26

    Li, S. J. & Hochstrasser, M. A new protease required for cell-cycle progression in yeast. Nature 398, 246–251 (1999).

  27. 27

    Jentsch, S. & Pyrowolakis, G. Ubiquitin and its kin: how close are the family ties? Trends Cell Biol. 10, 335–342 (2000).

  28. 28

    Rao, H., Uhlmann, F., Nasmyth, K. & Varshavsky, A. Degradation of a cohesin subunit by the N-end rule pathway is essential for chromosome stability. Nature 410, 955–959 (2001).

  29. 29

    Kannouche, P. et al. Domain structure, localization, and function of DNA polymerase η, defective in xeroderma pigmentosum variant cells. Genes Dev. 15, 158–172 (2001).

  30. 30

    Choi, J. H. & Pfeifer, G. P. The role of DNA polymerase η in UV mutational spectra. DNA Repair 4, 211–220 (2005).

  31. 31

    Parris, C. N., Levy, D. D., Jessee, J. & Seidman, M. M. Proximal and distal effects of sequence context on ultraviolet mutational hotspots in a shuttle vector replicated in xeroderma cells. J. Mol. Biol. 236, 491–502 (1994).

  32. 32

    Shi, Y. Caspase activation: revisiting the induced proximity model. Cell 117, 855–858 (2004).

  33. 33

    Matsuda, T., Bebenek, K., Masutani, C., Hanaoka, F. & Kunkel, T. A. Low fidelity DNA synthesis by human DNA polymerase η. Nature 404, 1011–1013 (2000).

  34. 34

    Zhu, Y. et al. DUB-2 is a member of a novel family of cytokine-inducible deubiquitinating enzymes. J. Biol. Chem. 272, 51–57 (1997).

  35. 35

    Vugmeyster, Y. et al. The ubiquitin-proteasome pathway in thymocyte apoptosis: caspase-dependent processing of the deubiquitinating enzyme USP7 (HAUSP). Mol. Immunol. 39, 431–441 (2002).

  36. 36

    Reiley, W. et al. Regulation of the deubiquitinating enzyme CYLD by IκB kinaseK-dependent phosphorylation. Mol. Cell Biol. 25, 3886–3895 (2005).

  37. 37

    Brummelkamp, T. R., Nijman, S. M., Dirac, A. M. & Bernards, R. Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF-κB. Nature 424, 797–801 (2003).

  38. 38

    Andreassen, P. R., D'Andrea, A. D. & Taniguchi, T. ATR couples FANCD2 monoubiquitination to the DNA-damage response. Genes Dev. 18, 1958–1963 (2004).

  39. 39

    Taniguchi, T. et al. S-phase-specific interaction of the Fanconi anemia protein, FANCD2, with BRCA1 and RAD51. Blood 100, 2414–2420 (2002).

Download references


We thank D. Finley and members of the D'Andrea laboratory for critical reading of the manuscript. We are grateful to V. Notenboom for technical assistance. We thank T. Taniguchi for RAD18 and PCNA cDNAs and A. Lehmann for GFP-polη expression constructs. We are grateful to M. M. Seidman for generously providing the pSP189 plasmid and the MBM7070 bacterial strain. This work was supported by grants from the National Institutes of Health (NIH, A.D.D.) and was funded in part by the Doris Duke Charitable Foundation (A.D.D.). T.T.H. is a Blount fellow of the Damon Runyon Cancer Research Foundation.

Author information

Correspondence to Alan D. D'Andrea.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures S1, S2, S3, S4 and S5 (PDF 115 kb)

Rights and permissions

Reprints and Permissions

About this article

Further reading