Fischer Jennifer A, Caradonna Salvatore
Department of Molecular Biology, University of Medicine and Dentistry, 08084, Stratford, NJ, USA.
Methods Mol Biol. 2011;761:137-49. doi: 10.1007/978-1-61779-182-6_9.
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.
尿嘧啶-DNA糖基化酶（UDG/UNG）是一类从DNA中去除尿嘧啶并启动碱基切除修复的酶。这些酶通过减少由G:C到A:T转换突变引起的诱变事件，在维持基因组完整性方面发挥关键作用。最近发现一类RNA编辑酶（AID/APOBECs）可使DNA中的胞嘧啶脱氨基，这引发了人们对这些碱基切除修复酶的兴趣。本文介绍的方法重点在于确定尿嘧啶-DNA糖基化酶核异构体（nUDG，一种36000道尔顿的蛋白质）的调控机制。在同步化的HeLa细胞中，nUDG蛋白水平在细胞周期的S期降至几乎检测不到的水平。当蛋白酶体依赖性蛋白质降解被阻断蛋白酶体的试剂抑制时，对免疫沉淀或亲和分离的nUDG进行免疫印迹分析，可揭示泛素缀合的nUDG。
