Cheng C, Shuman S
Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021 USA.
Nucleic Acids Res. 1997 Apr 1;25(7):1369-74. doi: 10.1093/nar/25.7.1369.
We report that Haemophilus influenzae encodes a 268 amino acid ATP-dependent DNA ligase. The specificity of Haemophilus DNA ligase was investigated using recombinant protein produced in Escherichia coli. The enzyme catalyzed efficient strand joining on a singly nicked DNA in the presence of magnesium and ATP (Km = 0.2 microM). Other nucleoside triphosphates or deoxynucleoside triphosphates could not substitute for ATP. Haemophilus ligase reacted with ATP in the absence of DNA substrate to form a covalent ligase-adenylate intermediate. This nucleotidyl transferase reaction required a divalent cation and was specific for ATP. The Haemophilus enzyme is the first example of an ATP-dependent DNA ligase encoded by a eubacterial genome. It is also the smallest member of the covalent nucleotidyl transferase superfamily, which includes the bacteriophage and eukaryotic ATP-dependent polynucleotide ligases and the GTP-dependent RNA capping enzymes.
我们报道了流感嗜血杆菌编码一种含268个氨基酸的ATP依赖性DNA连接酶。利用在大肠杆菌中产生的重组蛋白对流感嗜血杆菌DNA连接酶的特异性进行了研究。该酶在镁离子和ATP存在的情况下，能高效催化单切口DNA上的链连接反应（Km = 0.2微摩尔）。其他核苷三磷酸或脱氧核苷三磷酸不能替代ATP。流感嗜血杆菌连接酶在没有DNA底物的情况下与ATP反应，形成共价连接酶 - 腺苷酸中间体。这种核苷酸转移酶反应需要二价阳离子，且对ATP具有特异性。流感嗜血杆菌酶是真细菌基因组编码的ATP依赖性DNA连接酶的首个实例。它也是共价核苷酸转移酶超家族中最小的成员，该超家族包括噬菌体和真核生物的ATP依赖性多核苷酸连接酶以及GTP依赖性RNA加帽酶。
