Brown G W, Ray D S
Molecular Biology Institute, University of California, Los Angeles 90024.
Nucleic Acids Res. 1992 Aug 11;20(15):3905-10. doi: 10.1093/nar/20.15.3905.
A DNA ligase has been purified approximately 5000-fold, to near homogeneity, from the trypanosomatid Crithidia fasciculata. The purified enzyme contains polypeptides with molecular masses of 84 and 80 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both polypeptides formed enzyme-adenylate complexes in the absence of DNA, contained an epitope that is highly conserved between human and bovine DNA ligase I and yeast and vaccinia virus DNA ligases, and were identified in fresh lysates of C. fasciculata by antibodies raised against the purified protein. Hydrodynamic measurements indicate that the enzyme is an asymmetric protein of approximately 80 kDa. The purified DNA ligase can join oligo(dT) annealed to poly(dA), but not oligo(dT) annealed to poly(rA), and can ligate blunt-ended DNA fragments. The enzyme has a low Km for ATP of 0.3 microM. The DNA ligase absolutely requires ATP and Mg2+, and is inhibited by N-ethylmaleimide and by KCI. Substrate specificity, Km for ATP, and the conserved epitope all suggest that the purified enzyme is the trypanosome homologue of DNA ligase I.
已从锥虫克氏锥虫中纯化出一种DNA连接酶，纯化倍数约为5000倍，纯度接近均一。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳估计，纯化后的酶含有分子量分别为84 kDa和80 kDa的多肽。这两种多肽在无DNA的情况下均能形成酶-腺苷酸复合物，含有在人及牛DNA连接酶I、酵母和痘苗病毒DNA连接酶之间高度保守的表位，并且通过针对纯化蛋白产生的抗体在新鲜的克氏锥虫裂解物中得到鉴定。流体动力学测量表明，该酶是一种分子量约为80 kDa的不对称蛋白。纯化后的DNA连接酶能够连接与聚（dA）退火的寡聚（dT），但不能连接与聚（rA）退火的寡聚（dT），并且能够连接平端DNA片段。该酶对ATP的Km值较低，为0.3 μM。该DNA连接酶绝对需要ATP和Mg2+，并受到N-乙基马来酰亚胺和KCl的抑制。底物特异性、对ATP的Km值以及保守表位均表明，纯化后的酶是DNA连接酶I的锥虫同源物。
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