Skip to main content

Thank you for visiting 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.

RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO


The RAD6 pathway is central to post-replicative DNA repair in eukaryotic cells; however, the machinery and its regulation remain poorly understood. Two principal elements of this pathway are the ubiquitin-conjugating enzymes RAD6 and the MMS2–UBC13 heterodimer, which are recruited to chromatin by the RING-finger proteins RAD18 and RAD5, respectively. Here we show that UBC9, a small ubiquitin-related modifier (SUMO)-conjugating enzyme, is also affiliated with this pathway and that proliferating cell nuclear antigen (PCNA)—a DNA-polymerase sliding clamp involved in DNA synthesis and repair—is a substrate. PCNA is mono-ubiquitinated through RAD6 and RAD18, modified by lysine-63-linked multi-ubiquitination—which additionally requires MMS2, UBC13 and RAD5—and is conjugated to SUMO by UBC9. All three modifications affect the same lysine residue of PCNA, suggesting that they label PCNA for alternative functions. We demonstrate that these modifications differentially affect resistance to DNA damage, and that damage-induced PCNA ubiquitination is elementary for DNA repair and occurs at the same conserved residue in yeast and humans.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: PCNA is modified by SUMO in S. cerevisiae.
Figure 2: Regulation of PCNA SUMO modification and ubiquitination by DNA damage and link to the RAD6 pathway.
Figure 3: Role of PCNA modifications in DNA repair.
Figure 4: Human PCNA is ubiquitinated at the conserved K164 residue on DNA damage.
Figure 5: Model for ubiquitination and SUMO modification of PCNA.


  1. 1

    Prakash, S., Sung, P. & Prakash, L. DNA repair genes and proteins of Saccharomyces cerevisiae. Annu. Rev. Genet. 27, 33–70 (1993)

    CAS  Article  Google Scholar 

  2. 2

    Friedberg, E. C., Walker, G. C. & Siede, W. DNA Repair and Mutagenesis (American Society for Microbiology, Washington, 1995)

    Google Scholar 

  3. 3

    Hoeijmakers, J. H. Genome maintenance mechanisms for preventing cancer. Nature 411, 366–374 (2001)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Ulrich, H. D. Degradation or maintenance: actions of the ubiquitin system on eukaryotic chromatin. Eukaryotic Cell 1, 1–10 (2002)

    CAS  Article  Google Scholar 

  5. 5

    Jentsch, S., McGrath, J. P. & Varshavsky, A. The yeast DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme. Nature 329, 131–134 (1987)

    ADS  CAS  Article  Google Scholar 

  6. 6

    Broomfield, S., Chow, B. L. & Xiao, W. MMS2, encoding a ubiquitin-conjugating-enzyme-like protein, is a member of the yeast error-free postreplication repair pathway. Proc. Natl Acad. Sci. USA 95, 5678–5683 (1998)

    ADS  CAS  Article  Google Scholar 

  7. 7

    Hofmann, R. M. & Pickart, C. M. Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair. Cell 96, 645–653 (1999)

    CAS  Article  Google Scholar 

  8. 8

    Xiao, W. et al. Genetic interactions between error-prone and error-free postreplication repair pathways in Saccharomyces cerevisiae. Mutat. Res. 435, 1–11 (1999)

    CAS  Article  Google Scholar 

  9. 9

    Ulrich, H. D. & Jentsch, S. Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair. EMBO J. 19, 3388–3397 (2000)

    CAS  Article  Google Scholar 

  10. 10

    Moraes, T. F. et al. Crystal structure of the human ubiquitin conjugating enzyme complex, hMms2-hUbc13. Nature Struct. Biol. 8, 669–673 (2001)

    CAS  Article  Google Scholar 

  11. 11

    VanDemark, A. P., Hofmann, R. M., Tsui, C., Pickart, C. M. & Wolberger, C. Molecular insights into polyubiquitin chain assembly: crystal structure of the Mms2/Ubc13 heterodimer. Cell 105, 711–720 (2001)

    CAS  Article  Google Scholar 

  12. 12

    Bailly, V., Lamb, J., Sung, P., Prakash, S. & Prakash, L. Specific complex formation between yeast RAD6 and RAD18 proteins: a potential mechanism for targeting RAD6 ubiquitin-conjugating activity to DNA damage sites. Genes Dev. 8, 811–820 (1994)

    CAS  Article  Google Scholar 

  13. 13

    Pickart, C. M. Ubiquitin in chains. Trends Biochem. Sci. 25, 544–548 (2000)

    CAS  Article  Google Scholar 

  14. 14

    Melchior, F. SUMO-nonclassical ubiquitin. Annu. Rev. Cell Dev. Biol. 16, 591–626 (2000)

    CAS  Article  Google Scholar 

  15. 15

    Müller, S., Hoege, C., Pyrowolakis, G. & Jentsch, S. SUMO, ubiquitin's mysterious cousin. Nature Rev. Mol. Cell Biol. 2, 202–210 (2001)

    Article  Google Scholar 

  16. 16

    Hochstrasser, M. New structural clues to substrate specificity in the “ubiquitin system”. Mol. Cell 9, 453–454 (2002)

    CAS  Article  Google Scholar 

  17. 17

    Tsurimoto, T. PCNA binding proteins. Frontiers Biosci. 4, 849–858 (1999)

    Article  Google Scholar 

  18. 18

    Warbrick, E. The puzzle of PCNA's many partners. BioEssays 22, 997–1006 (2000)

    CAS  Article  Google Scholar 

  19. 19

    Bauer, G. A. & Burgers, P. M. Molecular cloning, structure and expression of the yeast proliferating cell nuclear antigen gene. Nucleic Acids Res. 18, 261–265 (1990)

    CAS  Article  Google Scholar 

  20. 20

    Johnson, E. S. & Blobel, G. Cell cycle-regulated attachment of the ubiquitin-related protein SUMO to the yeast septins. J. Cell Biol. 147, 981–994 (1999)

    CAS  Article  Google Scholar 

  21. 21

    Torres-Ramos, C. A., Yoder, B. L., Burgers, P. M., Prakash, S. & Prakash, L. Requirement of proliferating cell nuclear antigen in RAD6-dependent postreplicational DNA repair. Proc. Natl Acad. Sci. USA 93, 9676–9681 (1996)

    ADS  CAS  Article  Google Scholar 

  22. 22

    Spence, J., Sadis, S., Haas, A. L. & Finley, D. A ubiquitin mutant with specific defects in DNA repair and multiubiquitination. Mol. Cell. Biol. 15, 1265–1273 (1995)

    CAS  Article  Google Scholar 

  23. 23

    Ayyagari, R., Impellizzeri, K. J., Yoder, B. L., Gary, S. L. & Burgers, P. M. A mutational analysis of the yeast proliferating cell nuclear antigen indicates distinct roles in DNA replication and DNA repair. Mol. Cell. Biol. 15, 4420–4429 (1995)

    CAS  Article  Google Scholar 

  24. 24

    Joazeiro, C. A. & Weissman, A. M. RING finger proteins: mediators of ubiquitin ligase activity. Cell 102, 549–552 (2000)

    CAS  Article  Google Scholar 

  25. 25

    Bernier-Villamor, V., Sampson, D. A., Matunis, M. J. & Lima, C. D. Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitin-conjugating enzyme Ubc9 and RanGAP1. Cell 108, 345–356 (2002)

    CAS  Article  Google Scholar 

  26. 26

    Prakash, L. The RAD6 gene and protein of Saccharomyces cerevisiae. Ann. NY Acad. Sci. 726, 267–273 (1994)

    ADS  CAS  Article  Google Scholar 

  27. 27

    Johnson, R. E. et al. Saccharomyces cerevisiae RAD5-encoded DNA repair protein contains DNA helicase and zinc-binding sequence motifs and affects the stability of simple repetitive sequences in the genome. Mol. Cell. Biol. 12, 3807–3818 (1992)

    CAS  Article  Google Scholar 

  28. 28

    Lawrence, C. W. & Christensen, R. B. Metabolic suppressors of trimethoprim and ultraviolet light sensitivities of Saccharomyces cerevisiae rad6 mutants. J. Bacteriol. 139, 866–887 (1979)

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Schiestl, R. H., Prakash, S. & Prakash, L. The SRS2 suppressor of rad6 mutations of Saccharomyces cerevisiae acts by channeling DNA lesions into the RAD52 DNA repair pathway. Genetics 124, 817–831 (1990)

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Ulrich, H. D. The srs2 suppressor of UV sensitivity acts specifically on the RAD5- and MMS2-dependent branch of the RAD6 pathway. Nucleic Acids Res. 29, 3487–3494 (2001)

    CAS  Article  Google Scholar 

  31. 31

    Broomfield, S. & Xiao, W. Suppression of genetic defects within the RAD6 pathway by srs2 is specific for error-free post-replication repair but not for damage-induced mutagenesis. Nucleic Acids Res. 30, 732–739 (2002)

    CAS  Article  Google Scholar 

  32. 32

    Zhang, Z., Shibahara, K. & Stillman, B. PCNA connects DNA replication to epigenetic inheritance in yeast. Nature 408, 221–225 (2000)

    ADS  CAS  Article  Google Scholar 

  33. 33

    Krishna, T. S. et al. Crystallization of proliferating cell nuclear antigen (PCNA) from Saccharomyces cerevisiae. J. Mol. Biol. 241, 265–268 (1994)

    CAS  Article  Google Scholar 

  34. 34

    Amin, N. S. & Holm, C. In vivo analysis reveals that the interdomain region of the yeast proliferating cell nuclear antigen is important for DNA replication and DNA repair. Genetics 144, 479–493 (1996)

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Eissenberg, J. C., Ayyagari, R., Gomes, X. V. & Burgers, P. M. Mutations in yeast proliferating cell nuclear antigen define distinct sites for interaction with DNA polymerase δ and DNA polymerase ɛ. Mol. Cell. Biol. 17, 6367–6378 (1997)

    CAS  Article  Google Scholar 

  36. 36

    Johnson, E. S. & Gupta, A. A. An E3-like factor that promotes SUMO conjugation to the yeast septins. Cell 106, 735–744 (2001)

    CAS  Article  Google Scholar 

  37. 37

    Cejka, P., Vondrejs, V. & Storchova, Z. Dissection of the functions of the Saccharomyces cerevisiae RAD6 postreplicative repair group in mutagenesis and UV sensitivity. Genetics 159, 953–963 (2001)

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Xiao, W., Chow, B. L., Broomfield, S. & Hanna, M. The Saccharomyces cerevisiae RAD6 group is composed of an error-prone and two error-free postreplication repair pathways. Genetics 155, 1633–1641 (2000)

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Torres-Ramos, C. A., Prakash, S. & Prakash, L. Requirement of RAD5 and MMS2 for postreplication repair of UV-damaged DNA in Saccharomyces cerevisiae. Mol. Cell. Biol. 22, 2419–2426 (2002)

    CAS  Article  Google Scholar 

  40. 40

    Deng, L. et al. Activation of the IκB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell 103, 351–361 (2000)

    CAS  Article  Google Scholar 

  41. 41

    Wang, C. et al. TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 412, 346–351 (2001)

    ADS  CAS  Article  Google Scholar 

  42. 42

    Spence, J. et al. Cell cycle-regulated modification of the ribosome by a variant multiubiquitin chain. Cell 102, 67–76 (2000)

    CAS  Article  Google Scholar 

  43. 43

    Galan, J. M. & Haguenauer-Tsapis, R. Ubiquitin lys63 is involved in ubiquitination of a yeast plasma membrane protein. EMBO J. 16, 5847–5854 (1997)

    CAS  Article  Google Scholar 

  44. 44

    Brusky, J., Zhu, Y. & Xiao, W. UBC13, a DNA-damage-inducible gene, is a member of the error-free postreplication repair pathway in Saccharomyces cerevisiae. Curr. Genet. 37, 168–174 (2000)

    CAS  Article  Google Scholar 

  45. 45

    Tateishi, S., Sakuraba, Y., Masuyama, S., Inoue, H. & Yamaizumi, M. Dysfunction of human Rad18 results in defective postreplication repair and hypersensitivity to multiple mutagens. Proc. Natl Acad. Sci. USA 97, 7927–7932 (2000)

    ADS  CAS  Article  Google Scholar 

  46. 46

    Li, Z., Xiao, W., McCormick, J. J. & Maher, V. M. Identification of a protein essential for a major pathway used by human cells to avoid UV-induced DNA damage. Proc. Natl Acad. Sci. USA 99, 4459–4464 (2002)

    ADS  CAS  Article  Google Scholar 

  47. 47

    Desterro, J. M., Rodriguez, M. S. & Hay, R. T. SUMO-1 modification of IκBα inhibits NF-κB activation. Mol. Cell 2, 233–239 (1998)

    CAS  Article  Google Scholar 

  48. 48

    Seufert, W., Futcher, B. & Jentsch, S. Role of a ubiquitin-conjugating enzyme in degradation of S- and M-phase cyclins. Nature 373, 78–81 (1995)

    ADS  CAS  Article  Google Scholar 

  49. 49

    Knop, M. et al. Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines. Yeast 15, 963–972 (1999)

    CAS  Article  Google Scholar 

  50. 50

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

    ADS  CAS  Article  Google Scholar 

  51. 51

    Treier, M., Staszewski, L. M. & Bohmann, D. Ubiquitin-dependent c-Jun degradation in vivo is mediated by the δ domain. Cell 78, 787–798 (1994)

    CAS  Article  Google Scholar 

  52. 52

    Müller, S. et al. c-Jun and p53 activity is modulated by SUMO-1 modification. J. Biol. Chem. 275, 13321–13329 (2000)

    Article  Google Scholar 

Download references


We thank U. Cramer for technical assistance and S. Müller and M. Knop for experimental advice and discussions. We also thank P. Burgers, D. Finley, M. Hochstrasser, M. Knop, S. Müller and B. Stillman for plasmids and strains. S.J. is supported by the Max Planck Society, Deutsche Forschungsgemeinschaft and Fonds der chemischen Industrie.

Author information



Corresponding author

Correspondence to Stefan Jentsch.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


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