Sugino A, Goodman H M, Heyneker H L, Shine J, Boyer H W, Cozzarelli N R
J Biol Chem. 1977 Jun 10;252(11):3987-94.
The joining of duplex DNA at base-paired ends by bacteriophage T4 DNA ligase was confirmed using either a synthetic duplex decamer or restriction endonuclease fragments of ColE1 DNA as substrates. The reaction was not linearly dependent on enzyme concentration but increased markedly at high enzyme concentrations. Although T4 RNA ligase did not catalyze this blunt end joining, it makedly stimulated the DNA ligase reaction particularly at low DNA ligase concentrations. The apparent Km for the decamer was 50 micronM in the presence or absence of RNA ligase. In the presence of RNA ligase, T4 DNA ligase had about the same turnover number for blunt end and cohesive end joining. The joining of duplex DNA at base-paired ends was proven by several techniques including restriction endonuclease cleavage of the products. The products of the ligation reaction using restriction enzyme fragments were mostly linear oligomers but included some circular duplexes. Escherichia coli DNA ligase in the presence or absence of RNA ligase did not catalyze blunt end joining. RNA ligase only moderately affected the joining of cohesive ends by T4 DNA ligase or E. coli DNA ligase and did not itself catalyze this reaction.
利用合成的双链十聚体或大肠杆菌ColE1 DNA的限制性内切酶片段作为底物，证实了噬菌体T4 DNA连接酶能在碱基配对的末端连接双链DNA。该反应并非线性依赖于酶浓度，而是在高酶浓度下显著增加。虽然T4 RNA连接酶不催化这种平端连接，但它能显著刺激DNA连接酶反应，尤其是在低DNA连接酶浓度时。无论有无RNA连接酶，十聚体的表观Km值均为50微摩尔。在有RNA连接酶存在的情况下，T4 DNA连接酶平端连接和粘性末端连接的周转数大致相同。通过包括对产物进行限制性内切酶切割在内的几种技术，证实了双链DNA在碱基配对末端的连接。使用限制性酶切片段进行连接反应的产物大多是线性寡聚体，但也包括一些环状双链体。无论有无RNA连接酶，大肠杆菌DNA连接酶都不催化平端连接。RNA连接酶仅对T4 DNA连接酶或大肠杆菌DNA连接酶的粘性末端连接有适度影响，且其本身不催化该反应。
