Petersen B O, Shuman S
Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021, USA.
Nucleic Acids Res. 1997 Jun 1;25(11):2091-7. doi: 10.1093/nar/25.11.2091.
Vaccinia topoisomerase forms a covalent protein-DNA intermediate at sites containing the sequence 5'-CCCTT. The T nucleotide is linked via a 3'-phosphodiester bond to Tyr-274 of the enzyme. Here, we report that the enzyme catalyzes hydrolysis of the covalent intermediate, resulting in formation of a 3'-phosphate-terminated DNA cleavage product. The hydrolysis reaction is pH-dependent (optimum pH = 9.5) and is slower, by a factor of 10(-5), than the rate of topoisomerase-catalyzed strand transfer to a 5'-OH terminated DNA acceptor strand. Mutants of vaccinia topoisomerase containing serine or threonine in lieu of the active site Tyr-274 form no detectable covalent intermediate and catalyze no detectable DNA hydrolysis. This suggests that hydrolysis occurs subsequent to formation of the covalent protein-DNA adduct and not via direct attack by water on DNA. Vaccinia topoisomerase also catalyzes glycerololysis of the covalent intermediate. The rate of glycerololysis is proportional to glycerol concentration and is optimal at pH 9.5.
痘苗病毒拓扑异构酶在含有5'-CCCTT序列的位点形成共价蛋白-DNA中间体。T核苷酸通过3'-磷酸二酯键与该酶的Tyr-274相连。在此，我们报道该酶催化共价中间体的水解，导致形成3'-磷酸末端的DNA裂解产物。水解反应依赖于pH（最适pH = 9.5），并且比拓扑异构酶催化的链转移到5'-OH末端的DNA受体链的速率慢10^(-5)倍。用丝氨酸或苏氨酸取代活性位点Tyr-274的痘苗病毒拓扑异构酶突变体不形成可检测到的共价中间体，也不催化可检测到的DNA水解。这表明水解发生在共价蛋白-DNA加合物形成之后，而不是通过水直接攻击DNA。痘苗病毒拓扑异构酶还催化共价中间体的甘油解。甘油解的速率与甘油浓度成正比，在pH 9.5时最佳。
