Yang B, Wen X, Kodali N S, Oleykowski C A, Miller C G, Kulinski J, Besack D, Yeung J A, Kowalski D, Yeung A T
Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Biochemistry. 2000 Apr 4;39(13):3533-41. doi: 10.1021/bi992376z.
CEL I, isolated from celery, is the first eukaryotic nuclease known that cleaves DNA with high specificity at sites of base-substitution mismatch and DNA distortion. The enzyme requires Mg(2+) and Zn(2+) for activity, with a pH optimum at neutral pH. We have purified CEL I 33 000-fold to apparent homogeneity. A key improvement is the use of alpha-methyl-mannoside in the purification buffers to overcome the aggregation of glycoproteins with endogenous lectins. The SDS gel electrophoresis band for the homogeneous CEL I, with and without the removal of its carbohydrate moieties, was extracted, renatured, and shown to have mismatch cutting specificity. After determination of the amino acid sequence of 28% of the CEL I polypeptide, we cloned the CEL I cDNA. Potential orthologs are nucleases putatively encoded by the genes BFN1 of Arabidopsis, ZEN1 of Zinnia, and DSA6 of daylily. Homologies of CEL I with S1 and P1 nucleases are much lower. We propose that CEL I exemplifies a new family of neutral pH optimum, magnesium-stimulated, mismatch duplex-recognizing nucleases, within the S1 superfamily.
从芹菜中分离出的CEL I是已知的第一种真核核酸酶，它能在碱基取代错配和DNA扭曲位点以高特异性切割DNA。该酶的活性需要Mg(2+)和Zn(2+)，最适pH值为中性。我们已将CEL I纯化了33000倍，达到明显的均一性。一个关键的改进是在纯化缓冲液中使用α-甲基甘露糖苷，以克服糖蛋白与内源性凝集素的聚集。提取了去除和未去除碳水化合物部分的均一性CEL I的SDS凝胶电泳条带，使其复性，并显示出错配切割特异性。在确定了CEL I多肽28%的氨基酸序列后，我们克隆了CEL I cDNA。潜在的直系同源物是拟南芥的BFN1基因、百日草的ZEN1基因和萱草的DSA6基因推定编码的核酸酶。CEL I与S1和P1核酸酶的同源性要低得多。我们认为CEL I代表了S1超家族中一个新的家族，该家族具有最适中性pH值、镁刺激、错配双链识别核酸酶的特点。
