Ciarrocchi G, Lestingi M, Wright G, Montecucco A
Istituto di Genetica Biochimica ed Evoluzionistica, CNR, Pavia, Italy.
Nucleic Acids Res. 1993 Dec 25;21(25):5934-9. doi: 10.1093/nar/21.25.5934.
Both bacteriophage T4 and human type I DNA ligases in the presence of a mixture of ATP, AMP and PPi altered the topological properties of a supercoiled substrate by a step-wise reaction eventually leading to a population of fully relaxed, covalently closed products. In the presence of only AMP and PPi DNA products containing nicks with 3'OH/5'P termini accumulated in the presence of bacteriophage T4 DNA ligase, suggesting reversal of the entire joining reaction, but not in the presence of human DNA ligase I. Both DNA ligases became deoxyadenylylated in the presence of dATP, but the joining reaction did not proceed to completion. However, with both enzymes the full relaxing reaction took place in the presence of dAMP alone and in the presence of a mixture of dATP, dAMP and PPi. In no case could the joining reaction be reversed by dAMP and PPi. Related experiments with modified derivatives of deoxyribonucleoside 5'-triphosphates and PPi gave results in accord with these observations. The AMP dependent DNA relaxation catalysed by DNA ligases was insensitive to the presence of exonuclease III. These results indicate that controlled relaxation of the substrate by both DNA ligases occurs as a separate reaction rather than by simple reversal of the joining reaction. These findings support the hypothesis that in vivo the DNA topoisomerising ligases relax their substrate at the replication fork both during and separately from ligation of a pre-existing nick.
在ATP、AMP和焦磷酸（PPi）混合物存在的情况下，噬菌体T4和人类I型DNA连接酶都会通过逐步反应改变超螺旋底物的拓扑性质，最终产生一群完全松弛的共价闭合产物。在仅存在AMP和PPi的情况下，含有3'OH/5'P末端切口的DNA产物在噬菌体T4 DNA连接酶存在时会积累，这表明整个连接反应发生了逆转，但在人类DNA连接酶I存在时则不会。在dATP存在的情况下，两种DNA连接酶都会发生脱氧腺苷酸化，但连接反应并未进行到底。然而，对于这两种酶来说，在仅存在dAMP以及存在dATP、dAMP和PPi混合物的情况下，都会发生完全的松弛反应。在任何情况下，dAMP和PPi都不能使连接反应逆转。用脱氧核糖核苷5'-三磷酸和PPi的修饰衍生物进行的相关实验得到的结果与这些观察结果一致。DNA连接酶催化的依赖AMP的DNA松弛对外切核酸酶III的存在不敏感。这些结果表明，两种DNA连接酶对底物的可控松弛是作为一个独立反应发生的，而不是通过连接反应的简单逆转。这些发现支持了这样一种假说，即在体内，DNA拓扑异构化连接酶在复制叉处，在连接预先存在的切口的过程中以及与之分开的过程中，都会使其底物松弛。
