Definitive identification of mammalian 5-hydroxymethyluracil DNA N-glycosylase activity as SMUG1
Definitive identification of mammalian 5-hydroxymethyluracil DNA N-glycosylase activity as SMUG1
复制标题
DOI：
10.1074/jbc.m106953200
复制
发表时间：
2001-11-09
影响因子：
4.8
通讯作者：
Teebor, GW
中科院分区：
文献类型：
--
作者：
Boorstein, RJ;Cummings, A;Teebor, GW
从小牛胸腺中纯化了从枯草芽孢杆菌噬菌体SPO1 DNA中释放5-羟甲基尿嘧啶（5hmUra）的DNA n -糖基酶活性（HMUDG）。用sds -聚丙烯酰胺凝胶电泳对纯化部分进行分析，发现蛋白质种类繁多，无法通过检查鉴定HMUDG。因此，我们在sds -聚丙烯酰胺凝胶电泳后对酶进行了再生，并对凝胶切片进行了针对5hmUra的DNA n -糖基酶活性测定。酶活性在分子质量标记30 ~ 34 kDa之间最大。从凝胶切片中提取蛋白质，进行胰蛋白酶消化和质谱分析。分析结果显示，有11个肽段与最近鉴定的人类单链单功能尿嘧啶DNA n -糖基化酶（hSMUG1）序列同源或相同。hSMUG1的cDNA被分离并表达为重组谷胱甘肽s -转移酶融合蛋白，其释放5hmUra的比活性是最纯化的牛部分的20倍。我们得出结论，hSMUG1和HMUDG是相同的蛋白。
Purification from calf thymus of a DNA N-glycosylase activity (HMUDG) that released 5-hydroxymethyluracil (5hmUra) from the DNA of Bacillus subtilis phage SPO1 was undertaken. Analysis of the most purified fraction by SDS-polyacrylamide gel electrophoresis revealed a multiplicity of protein species making it impossible to identify HMUDG by inspection. Therefore, we renatured the enzyme after SDS-polyacrylamide gel electrophoresis and assayed slices of the gel for DNA N-glycosylase activity directed against 5hmUra. Maximum enzymatic activity was identified between molecular mass markers 30 and 34 kDa. Protein was extracted from gel slices and subjected to tryptic digestion and analysis by mass spectrometry. Analysis revealed the presence of 11 peptides that were homologous or identical to the sequence of the recently characterized human single-stranded monofunctional uracil DNA N-glycosylase (hSMUG1). The cDNA of hSMUG1 was isolated and expressed as a recombinant glutathione S-transferase fusion protein that was shown to release 5hmUra with 20X the specific activity of the most purified bovine fraction. We conclude that hSMUG1 and HMUDG are the same protein.