Robins P, Lindahl T
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, United Kingdom.
J Biol Chem. 1996 Sep 27;271(39):24257-61. doi: 10.1074/jbc.271.39.24257.
A human cDNA encoding a previously unrecognized DNA ligase IV has been identified (Wei, Y.-F., Robins, P., Carter, K., Caldecott, K., Pappin, D. J. C., Yu, G.-L., Wang, R.-P., Shell, B. K., Nash, R. A., Schär, P., Barnes, D. E., Haseltine, W. A., and Lindahl, T. (1995) Mol. Cell. Biol. 15, 3206-3216). Antibodies have been raised against predicted peptide sequences of DNA ligase IV and used to identify the enzyme during purification from HeLa cell nuclei. The 96-kDa DNA ligase IV and the 103-kDa DNA ligase III co-migrate during SDS-polyacrylamide gel electrophoresis and have similar column fractionation properties, which complicates the distinction between the two enzymes, but they have been separated by Mono S liquid chromatography. During initial size fractionation by gel chromatography in 1 M NaCl, DNA ligase IV elutes in the same position as the DNA ligase III-XRCC1 protein complex, indicating that DNA ligase IV is also bound to another protein or occurs as a dimer. DNA ligase IV has been purified free from other DNA ligases, and its enzymatic properties have been examined. The purified protein effectively joins single-strand breaks in a double-stranded polydeoxynucleotide in an ATP-dependent reaction. The substrate specificity of DNA ligase IV differs from those of the other two cloned human DNA ligases, I and III, with regard to their ability to join the hybrid substrates oligo(dT).poly(rA) and oligo(rA).poly(dT). DNA ligase IV occurs in part as an enzyme-adenylate complex in HeLa cell nuclear extracts.
已鉴定出一种编码此前未被识别的DNA连接酶IV的人类cDNA（魏，Y.-F.，罗宾斯，P.，卡特，K.，卡尔德科特，K.，帕平，D.J.C.，于，G.-L.，王，R.-P.，谢尔，B.K.，纳什，R.A.，沙尔，P.，巴恩斯，D.E.，哈塞尔廷，W.A.，和林达尔，T.（1995年）《分子细胞生物学》15，3206 - 3216）。针对DNA连接酶IV的预测肽序列制备了抗体，并用于在从HeLa细胞核中纯化该酶的过程中鉴定该酶。96 kDa的DNA连接酶IV和103 kDa的DNA连接酶III在SDS - 聚丙烯酰胺凝胶电泳过程中共迁移，并且具有相似的柱分级分离特性，这使得区分这两种酶变得复杂，但它们已通过单磺酸（Mono S）液相色谱法分离。在1 M NaCl中通过凝胶色谱进行初始大小分级分离时，DNA连接酶IV与DNA连接酶III - XRCC1蛋白复合物在相同位置洗脱，表明DNA连接酶IV也与另一种蛋白质结合或作为二聚体存在。已将DNA连接酶IV从其他DNA连接酶中纯化出来，并对其酶学性质进行了研究。纯化后的蛋白质在ATP依赖性反应中有效地连接双链多脱氧核苷酸中的单链断裂。就连接杂交底物寡聚（dT）·聚（rA）和寡聚（rA）·聚（dT）的能力而言，DNA连接酶IV的底物特异性不同于另外两种已克隆的人类DNA连接酶I和III。在HeLa细胞核提取物中，DNA连接酶IV部分以酶 - 腺苷酸复合物的形式存在。
