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

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

Abstract

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 

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

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

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