Analysis of Nuclear Uracil–DNA Glycosylase (nUDG) Turnover During the Cell Cycle

  • Jennifer A. FischerEmail author
  • Salvatore Caradonna
Part of the Methods in Molecular Biology book series (MIMB, volume 761)


Uracil–DNA glycosylases (UDG/UNG) are enzymes that remove uracil from DNA and initiate base-excision repair. These enzymes play a key role in maintaining genomic integrity by reducing the mutagenic events caused by G:C to A:T transition mutations. The recent finding that a family of RNA editing enzymes (AID/APOBECs) can deaminate cytosine in DNA has raised the interest in these base-excision repair enzymes. The methodology presented here focuses on determining the regulation of the nuclear isoform of uracil–DNA glycosylase (nUDG), a 36,000 Da protein. In synchronized HeLa cells, nUDG protein levels decrease to barely detectable levels during the S phase of the cell cycle. Immunoblot analysis of immunoprecipitated or affinity-isolated nUDG reveals ubiquitin-conjugated nUDG when proteolysis is inhibited by agents that block proteasomal-dependent protein degradation.

Key words

Uracil–DNA glycosylase cell cycle ubiquitin conjugation immunoprecipitation cell transfection 


  1. 1.
    Lindahl, T., and Wood, R. D. (1999) Quality control by DNA repair. Science 286, 1897–1905.PubMedCrossRefGoogle Scholar
  2. 2.
    Pearl, L. H. (2000) Structure and function in the uracil-DNA glycosylase superfamily. Mutat. Res. 460, 165–181.PubMedGoogle Scholar
  3. 3.
    Krokan, H. E., Standal, R., and Slupphaug, G. (1997) DNA glycosylases in the base excision repair of DNA. Biochem. J. 325, 1–16.PubMedGoogle Scholar
  4. 4.
    Rada, C., Williams, G. T., Nilsen, H., Barnes, D. E., Lindahl, T., and Neuberger, M. S. (2002) Immunoglobulin isotype switching is inhibited and somatic hypermutation perturbed in UNG-deficient mice. Curr. Biol. 12, 1748–1755.PubMedCrossRefGoogle Scholar
  5. 5.
    Di Noia, J., and Neuberger, M. S. (2002) Altering the pathway of immunoglobulin hypermutation by inhibiting uracil-DNA glycosylase. Nature 419, 43–48.PubMedCrossRefGoogle Scholar
  6. 6.
    Harris, R. S., Sheehy, A. M., Craig, H. M., Petersen-Mahrt, S. K., Watt, I. N., Neuberger, M. S., and Malim, M. H. (2003) DNA deamination mediates innate immunity to retroviral infection. Cell 113, 803–809.PubMedCrossRefGoogle Scholar
  7. 7.
    Fischer, J. A., Muller-Weeks, S., and Caradonna, S. (2004) Proteolytic degradation of the nuclear isoform of uracil-DNA glycosylase occurs during the S phase of the cell cycle. DNA Repair (Amst) 3, 505–513.CrossRefGoogle Scholar
  8. 8.
    Fischer, J. A., Muller-Weeks, S., and Caradonna, S. J. (2006) Fluorodeoxyuridine modulates cellular expression of the DNA base excision repair enzyme uracil-DNA glycosylase. Cancer Res. 66, 8829–8837.PubMedCrossRefGoogle Scholar
  9. 9.
    Treier, M., Staszewski, L. M., and Bohmann, D. (1994) Ubiquitin-dependent c-Jun degradation in vivo is mediated by the delta domain. Cell 78, 787–798.PubMedCrossRefGoogle Scholar
  10. 10.
    Salghetti, S. E., Kim, S. Y., and Tansey, W. P. (1999) Destruction of Myc by ubiquitin-mediated proteolysis: cancer-associated and transforming mutations stabilize Myc. EMBO J. 18, 717–726.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Molecular BiologyUniversity of Medicine and DentistryStratfordUSA

Personalised recommendations