Tse-Dinh Y C
J Biol Chem. 1986 Aug 15;261(23):10931-5.
DNA topoisomerases have been shown to cleave DNA phosphodiester bond and simultaneously become linked to the DNA at the cleavage site via a phosphotyrosine linkage (Tse, Y.-C., Kirkegaard, K., and Wang, J. C. (1980) J. Biol. Chem. 255, 5560-5565). For prokaryotic DNA topoisomerases, this is observed only when denaturant or protease is added to the topoisomerase-DNA incubation mixture. Previous attempts to reform DNA phosphodiester bonds from the covalent protein-DNA complex have been unsuccessful. Using oligonucleotides as substrates, the cleavage reaction of Escherichia coli DNA topoisomerase I occurs spontaneously (Tse-Dinh, Y.-C., McCarron, B. G. H., Arentzen, R., and Chowdhry, V. (1983) Nucleic Acids Res. 11, 8691-8701). Upon reaction with oligo(dA) labeled with 32P using terminal transferase and [alpha-32P]dATP, the enzyme becomes covalently linked to the 32P-labeled oligonucleotide. This 32P label can then be transferred to the 3'-OH end of a linear or nicked duplex DNA molecule subsequently added to the reaction mixture. This phosphodiester bond rejoining reaction can occur at a recessed, blunt, or protruding 3'-end of double-stranded DNA. It requires magnesium ions. These observations suggest that the covalent protein-DNA complex is a true intermediate during topoisomerization. Implications on the structure of prokaryotic type I DNA topoisomerases as compared to their eukaryotic counterparts are discussed.
已证明DNA拓扑异构酶可切割DNA磷酸二酯键，并同时通过磷酸酪氨酸连接在切割位点与DNA相连（谢育成、柯克加德、王中诚，《生物化学杂志》，1980年，第255卷，第5560 - 5565页）。对于原核生物DNA拓扑异构酶，只有在向拓扑异构酶 - DNA孵育混合物中加入变性剂或蛋白酶时才会观察到这种情况。此前尝试从共价蛋白 - DNA复合物中重新形成DNA磷酸二酯键均未成功。以寡核苷酸为底物时，大肠杆菌DNA拓扑异构酶I的切割反应会自发发生（谢 - 丁育成、麦卡伦、阿伦岑、乔德里，《核酸研究》，1983年，第11卷，第8691 - 8701页）。使用末端转移酶和[α - 32P]dATP对用32P标记的寡聚（dA）进行反应后，该酶会与32P标记的寡核苷酸共价连接。然后，这个32P标记可转移到随后添加到反应混合物中的线性或带切口双链DNA分子的3'-OH末端。这种磷酸二酯键重新连接反应可发生在双链DNA的凹陷、平端或突出3'-末端。它需要镁离子。这些观察结果表明，共价蛋白 - DNA复合物是拓扑异构化过程中的真正中间体。文中还讨论了与真核生物对应物相比原核生物I型DNA拓扑异构酶的结构含义。
