Purification of a mammalian homologue of Escherichia coli endonuclease III: Identification of a bovine pyrimidine hydrate thymine glycol DNA-glycosylase AP lyase by irreversible cross linking to a thymine glycol-containing oligodeoxynucleotide
Purification of a mammalian homologue of Escherichia coli endonuclease III: Identification of a bovine pyrimidine hydrate thymine glycol DNA-glycosylase AP lyase by irreversible cross linking to a thymine glycol-containing oligodeoxynucleotide
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DOI：
10.1021/bi952516e
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发表时间：
1996-02-27
期刊：
影响因子：
2.9
通讯作者：
Teebor, GW
中科院分区：
文献类型：
--
作者：
Hilbert, TP;Boorstein, RJ;Teebor, GW
我们从小牛胸腺中纯化了大肠杆菌DNA修复酶内切酶III的同源物，该同源物与内切酶III一样，具有抗嘧啶水合物和胸腺嘧啶乙二醇的DNA糖基酶活性和AP裂解酶活性（通过β -消除在AP位点切割DNA链）。酶之间的功能相似性表明，基于其酶底物（ES）中间体的性质，可以确定牛蛋白的鉴定策略。原核DNA糖基化酶/AP裂解酶通过n -酰基胺（希夫斯碱）ES中间体起作用，这些中间体在化学还原为稳定的仲胺后，不可逆地将酶交联到含有底物修饰碱基的寡脱氧核苷酸上。我们在NaCNBH3存在下，用p -32标记的含胸腺嘧啶二醇的寡脱氧核苷酸孵育内切酶III。这导致酶的SDS-PAGE表观分子量增加。磷酸化成像证实了酶和DNA之间的不可逆交联，对大多数纯化的牛酶部分进行相同处理导致寡脱氧核苷酸与优势的31 kDa物种不可逆交联，31 kDa物种的氨基酸分析显示与秀丽隐杆线虫27.8 kDa蛋白的预测氨基酸序列同源，而后者又与内切酶III具有同源性。从智人（Homo sapiens）和家鼠（Rattus sp）中翻译的两个部分3' cdna的氨基酸序列也与秀丽隐杆线虫和牛的序列具有同源性，这表明在整个系统发育过程中存在同源的核酸内切酶iii样DNA修复酶家族。
We purified a homologue of the Escherichia coli DNA repair enzyme endonuclease III 5000-fold from calf thymus which, like endonuclease III, demonstrates DNA-glycosylase activity against pyrimidine hydrates and thymine glycol and AP lyase activity (DNA strand cleavage at AP sites via beta-elimination). The functional similarity between the enzymes suggested a strategy for definitive identification of the bovine protein based on the nature of its enzyme-substrate (ES) intermediate. Prokaryotic DNA glycosylase/AP lyases function through N-acylimine (Schiffs base) ES intermediates which, upon chemical reduction to stable secondary amines, irreversibly cross link the enzyme to oligodeoxynucleotides containing substrate modified bases. We incubated endonuclease III with a P-32-labeled thymine glycol-containing oligodeoxynucleotide in the presence of NaCNBH3. This resulted in an increase in the apparent molecular weight of the enzyme by SDS-PAGE. Phosphorimaging confirmed irreversible cross linking between enzyme and DNA, Identical treatment of the most purified bovine enzyme fraction resulted in irreversible cross linking of the oligodeoxynucleotide to a predominant 31 kDa species, Amino acid analysis of the 31 kDa species revealed homology to the predicted amino acid sequence of a Caenorhabditis elegans 27.8 kDa protein which, in turn, has homology to endonuclease III. The translated amino acid sequences of two partial 3' cDNAs, from Homo sapiens and Rattus sp,, also demonstrate homology to the C. elegans and bovine sequences suggesting a homologous family of endonuclease III-like DNA repair enzymes is present throughout phylogeny.