Analysis of Nuclear Uracil DNA-Glycosylase (nUDG) Turnover During the Cell Cycle.
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.
Department of Molecular Biology, University of Medicine and Dentistry of New Jersey, Stratford, NJ, USA.
Jennifer A
JA
Fischer
Department of Molecular Biology, Rowan School of Osteopathic Medicine, University of Medicine and Dentistry of New Jersey, Two Medical Center Drive, Stratford, NJ, 08084, USA. caradonn@rowan.edu.
Salvatore J
SJ
Caradonna
D015153:Blotting, Western; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology*; D002467:Cell Nucleus / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D025801:Ubiquitin / Q000378:metabolism; D051981:Uracil-DNA Glycosidase / Q000235:genetics / Q000378:metabolism*
D025801:Ubiquitin; D051981:Uracil-DNA Glycosidase
Cell cycle; Cell transfection; Immunoprecipitation; Ubiquitin conjugation; Uracil-DNA glycosylase