Takahashi M, Yamaguchi E, Uchida T
J Biol Chem. 1984 Aug 25;259(16):10041-7.
Thermophilic and thermostable DNA ligase was purified to near homogeneity from the extract of Thermus thermophilus HB8. The purified enzyme has an isoelectric point at pH 6.6 and consists of a single polypeptide of about 79,000 in molecular weight on the bases of sodium dodecyl sulfate-polyacrylamide gel electrophoresis data and an equilibrium sedimentation method. The enzyme requires divalent cations, Mg2+ or Mn2+, and the optimum concentration of these ions being 5-9 X 10(-3) M and 3-6 X 10(-3) M, respectively. The enzyme also requires NAD as a cofactor. The apparent Km for NAD is 1.85 X 10(-8) M and that of (dT)10 is 1.4 X 10(-4) M. The pH optimum is 7.4-7.6 in Tris-HCl and 8.0 in collidine/HCl buffer. The joining reaction is activated by K+ and NH+4 at a concentration of 2-100 mM and inhibited by Na+ above 25 mM. The optimum temperatures of the joining of thymidylate oligomers in the presence of poly(dA) as a template are 27.5 degrees C for p(dT)s, 34.5 degrees C for p(dT)10, and 37 degrees C for p(dT)12-18 and that of cohesive-end DNA restriction fragments is 24-37 degrees C. The nick-closing activity of the enzyme was observed over a wide range of the temperature from 15 to 85 degrees C and the optimum temperature is 65-72 degrees C. The temperature dependency of ligation with HB8 DNA ligase for various substrates was found to shift to a region of 7-10 degrees C higher than that of T4 DNA ligase and the activity of HB8 DNA ligase decreased remarkably below 4 degrees C. The enzyme was stable for 1 week at 37 degrees C, its activity dropped by 50% within 2 days at 65 degrees C.
嗜热且耐热的DNA连接酶从嗜热栖热菌HB8提取物中纯化至近乎同质。根据十二烷基硫酸钠-聚丙烯酰胺凝胶电泳数据和平衡沉降法，纯化后的酶在pH 6.6处有一个等电点，由一条分子量约为79,000的单链多肽组成。该酶需要二价阳离子Mg2+或Mn2+，这些离子的最佳浓度分别为5 - 9×10(-3)M和3 - 6×10(-3)M。该酶还需要NAD作为辅因子。NAD的表观Km为1.85×10(-8)M，(dT)10的表观Km为1.4×10(-4)M。在Tris-HCl中最适pH为7.4 - 7.6，在可力丁/盐酸缓冲液中为8.0。连接反应在2 - 100 mM浓度的K+和NH4+作用下被激活，在25 mM以上的Na+存在下受到抑制。以聚(dA)为模板时，胸苷酸寡聚物连接的最适温度，对于p(dT)s为27.5℃，对于p(dT)10为34.5℃，对于p(dT)12 - 18为37℃，粘性末端DNA限制片段连接的最适温度为24 - 37℃。在15至85℃的宽温度范围内观察到该酶的切口封闭活性，最适温度为65 - 72℃。发现HB8 DNA连接酶对各种底物的连接温度依赖性比T4 DNA连接酶向高7 - 10℃的区域偏移，并且HB8 DNA连接酶的活性在4℃以下显著下降。该酶在37℃下可稳定保存1周，在65℃下2天内活性下降50%。
