Molecular characterization of a human DNA kinase
Molecular characterization of a human DNA kinase
复制标题
DOI：
10.1074/jbc.274.34.24187
复制
发表时间：
1999-08-20
影响因子：
4.8
通讯作者：
Weinfeld, M
中科院分区：
文献类型：
--
作者：
Karimi-Busheri, F;Daly, G;Weinfeld, M
人多聚脱氧核糖核苷酸激酶是一种能够在5 ′-羟基末端磷酸化DNA并使3 ′-磷酸末端去磷酸化的酶，因此，可以认为是一种推定的DNA修复酶。从HeLa细胞中纯化该酶。用质谱法测定了几个胰蛋白酶片段的氨基酸序列，通过dbEST数据库与一个不完整的人cDNA克隆进行比对，并以该cDNA克隆为探针从一个单独的cDNA文库中检索cDNA序列的5'端。在大肠杆菌中表达了编码521个氨基酸（57.1 kDa）的蛋白质的完整cDNA，重组蛋白具有激酶和磷酸酶活性。与其他测序的蛋白质比较，鉴定了一个P-环基序，指示ATP结合结构域，以及与几种不同磷酸酶相关的第二基序。与秀丽隐杆线虫和粟酒裂殖酵母基因组中推定的开放阅读框有合理的序列相似性，但与噬菌体T4多核苷酸激酶的相似性限于上述激酶和磷酸酶结构域。北方杂交显示约2.3种酶的主要转录物和约7种酶的次要转录物。胰腺、心脏和肾脏的mRNA水平似乎高于脑、肺或肝脏。人A549细胞的共聚焦显微镜表明，激酶主要存在于细胞核中。编码该酶的基因定位于染色体19q13.4带。
Human polydeoxyribonucleotide kinase is an enzyme that has the capacity to phosphorylate DNA at 5'-hydroxyl termini and dephosphorylate 3'-phosphate termini and, therefore, can be considered a putative DNA repair enzyme. The enzyme was purified from HeLa cells. Amino acid sequence was obtained for several tryptic fragments by mass spectrometry, The sequences were matched through the dbEST data base with an incomplete human cDNA clone, which was used as a probe to retrieve the 5' end of the cDNA sequence from a separate cDNA library. The complete cDNA, which codes for a 521-amino acid protein (57.1 kDa), was expressed in Escherichia coli, and the recombinant protein was shown to possess the kinase and phosphatase activities. Comparison with other sequenced proteins identified a P-loop motif, indicative of an ATP-binding domain, and a second motif associated with several different phosphatases, There is reasonable sequence similarity to putative open reading frames in the genomes of Caenorhabditis elegans and Schizosaccharomyces pombe, but similarity to bacteriophage T4 polynucleotide kinase is limited to the kinase and phosphatase domains noted above. Northern hybridization revealed a major transcript of approximately 2.3 kilobases and a minor transcript of approximately 7 kilobases, Pancreas, heart, and kidney appear to have higher levels of mRNA than brain, lung, or liver. Confocal microscopy of human A549 cells indicated that the kinase resides predominantly in the nucleus. The gene encoding the enzyme was mapped to chromosome band 19q13.4.