Proteolytic degradation of the nuclear isoform of uracil-DNA glycosylase occurs during the S phase of the cell cycle
Proteolytic degradation of the nuclear isoform of uracil-DNA glycosylase occurs during the S phase of the cell cycle
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DOI：
10.1016/j.dnarep.2004.01.012
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发表时间：
2004-05-04
期刊：
影响因子：
3.8
通讯作者：
Caradonna, S
中科院分区：
文献类型：
--
作者：
Fischer, JA;Muller-Weeks, S;Caradonna, S
尿嘧啶-DNA糖基化酶是从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，一个家族的RNA编辑酶（APOBECs）可以脱氨基DNA中的胞嘧啶引起了人们对这些碱基切除修复酶的兴趣。这项研究的重点是尿嘧啶-DNA糖基化酶，一个36,000 Da的蛋白质，含有一个独特的44个氨基酸的N-末端的核亚型的调控。在同步化的HeLa细胞中，UDG 1A蛋白水平在细胞周期的S期下降到几乎检测不到的水平。免疫沉淀或亲和分离的UDG 1A的免疫印迹分析显示，当使用N-乙酰基-leu-teu-norleu-al或MG 132（蛋白体依赖性蛋白质降解的抑制剂）抑制蛋白水解时，UDG 1A为泛素结合的。使用组氨酸标记的泛素进行瞬时转染实验，以确认内源性UDG 1A在体内被泛素化。加入核输出抑制剂来普霉素B可防止UDG 1A的泛素化和降解。这表明从细胞核的易位可能是UDG 1A周转的一个步骤。最后，当细胞与细胞周期蛋白依赖性激酶抑制剂roscovitine孵育时，UDG 1A蛋白降解被阻止。这些结果表明，蛋白磷酸化和/或核输出参与UDG 1A蛋白水平的翻译后调节。(C)2004 Elsevier B. V.保留所有权利。
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 36,000 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-teu-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. (C) 2004 Elsevier B.V. All rights reserved.