Definitive identification of mammalian 5-hydroxymethyluracil DNA N-glycosylase activity as SMUG1

Purification from calf thymus of a DNA N-glycosylase activity (HMUDG) that released 5-hydroxymethyluracil (5hmUra) from the DNA of Bacillus subtilis p hage SPO1 was undertaken. Analysis of the most purified fraction by SDS-pol yacrylamide gel electrophoresis revealed a multiplicity of protein species making it impossible to identify HMUDG by inspection. Therefore, we renatur ed the enzyme after SDS-polyacrylamide gel electrophoresis and assayed slic es of the gel for DNA N-glycosylase activity directed against 5hmUra. Maxim um enzymatic activity was identified between molecular mass markers 30 and 34 kDa. Protein was extracted from gel slices and subjected to tryptic dige stion and analysis by mass spectrometry. Analysis revealed the presence of 11 peptides that were homologous or identical to the sequence of the recent ly characterized human single-stranded monofunctional uracil DNA N-glycosyl ase (hSMUG1). The cDNA of hSMUG1 was isolated and expressed as a recombinan t glutathione S-transferase fusion protein that was shown to release 5hmUra with 20X the specific activity of the most purified bovine fraction. We co nclude that hSMUG1 and HMUDG are the same protein.