Mammalian DNA ligases. Catalytic domain and size of DNA ligase I.
Mammalian DNA ligases. Catalytic domain and size of DNA ligase I.
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哺乳动物 DNA 连接酶。
DOI：
10.1016/s0021-9258(19)38387-5
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
T. Lindahl
中科院分区：
文献类型：
--
作者：
A. Tomkinson;D. Lasko;G. Daly;T. Lindahl
DNA连接酶I是增殖哺乳动物细胞中主要的DNA连接酶活性。该蛋白质已从小牛胸腺中纯化至表观均一性。它具有单体结构和封闭的N-末端残基。DNA连接酶I是一种125 kDa的多肽，通过十二烷基硫酸钠凝胶电泳和变性条件下的凝胶色谱法估计，而流体动力学测量表明，该酶是一种不对称的98 kDa的蛋白质。兔多克隆抗体的酶的免疫印迹显示一个单一的多肽125 kDa的新鲜制备的小牛胸腺粗细胞提取物。用几种试剂蛋白水解酶对纯化的DNA连接酶I进行有限消化，产生相对蛋白酶抗性的85-kDa片段。该结构域保留了完整的催化活性。用部分纯化的人DNA连接酶I获得了类似的结果。活性大片段代表完整蛋白质的C-末端部分，并且含有哺乳动物DNA连接酶I与酵母和牛痘病毒DNA连接酶之间保守的表位。DNA连接酶I的N-末端区域的功能是未知的。
DNA ligase I is the major DNA ligase activity in proliferating mammalian cells. The protein has been purified to apparent homogeneity from calf thymus. It has a monomeric structure and a blocked N-terminal residue. DNA ligase I is a 125-kDa polypeptide as estimated by sodium dodecyl sulfate-gel electrophoresis and by gel chromatography under denaturing conditions, whereas hydrodynamic measurements indicate that the enzyme is an asymmetric 98-kDa protein. Immunoblotting with rabbit polyclonal antibodies to the enzyme revealed a single polypeptide of 125 kDa in freshly prepared crude cell extracts of calf thymus. Limited digestion of the purified DNA ligase I with several reagent proteolytic enzymes generated a relatively protease-resistant 85-kDa fragment. This domain retained full catalytic activity. Similar results were obtained with partially purified human DNA ligase I. The active large fragment represents the C-terminal part of the intact protein, and contains an epitope conserved between mammalian DNA ligase I and yeast and vaccinia virus DNA ligases. The function of the N-terminal region of DNA ligase I is unknown.