Proteolytic degradation of the nuclear isoform of uracil-DNA glycosylase occurs during the S phase of the cell cycle.
Uracil-DNA glycosylases 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 (APOBECs) can deaminate cytosine in DNA has raised the interest in these base-excision repair enzymes. This research focuses on the regulation of the nuclear isoform of uracil-DNA glycosylase, a 36000 Da protein that contains a unique 44 amino acid N-terminus. In synchronized HeLa cells, UDG1A protein levels decrease to barely detectable levels during the S phase of the cell cycle. Immunoblot analysis of immunoprecipitated or affinity-isolated UDG1A reveals ubiquitin-conjugated UDG1A when proteolysis is inhibited using N-acetyl-leu-leu-norleu-al or MG132, inhibitors of proteosomal dependent protein degradation. Transient transfection experiments, with histidine-tagged ubiquitin, were used to confirm that endogenous UDG1A is ubiquitinated in vivo. Addition of the nuclear export inhibitor, leptomycin B, prevents ubiquitination and degradation of UDG1A. This indicates that translocation from the nucleus may be a step in UDG1A turnover. Finally, UDG1A protein degradation is prevented when cells are incubated with the cyclin-dependent kinase inhibitor, roscovitine. These results suggest that protein phosphorylation and/or nuclear export participate in the post-translational regulation of UDG1A protein levels.
Fischer
Jennifer A
JA
Department of Molecular Biology, Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey, Two Medical Center Drive, Stratford, NJ 08084, USA.
Muller-Weeks
Susan
S
Caradonna
Sal
S
D002467:Cell Nucleus / Q000201:enzymology*; D045647:DNA Glycosylases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D010766:Phosphorylation; D016196:S Phase; D025801:Ubiquitin / Q000378:metabolism; D051981:Uracil-DNA Glycosidase
D007527:Isoenzymes; D025801:Ubiquitin; D045647:DNA Glycosylases; D051981:Uracil-DNA Glycosidase