Huang You-Yi, Deng Jiao-Yu, Gu Jing, Zhang Zhi-Ping, Maxwell Anthony, Bi Li-Jun, Chen Yuan-Yuan, Zhou Ya-Feng, Yu Zi-Niu, Zhang Xian-En
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
Nucleic Acids Res. 2006;34(19):5650-9. doi: 10.1093/nar/gkl695. Epub 2006 Oct 11.
As only the type II topoisomerase is capable of introducing negative supercoiling, DNA gyrase is involved in crucial cellular processes. Although the other domains of DNA gyrase are better understood, the mechanism of DNA binding by the C-terminal domain of the DNA gyrase A subunit (GyrA-CTD) is less clear. Here, we investigated the DNA-binding sites in the GyrA-CTD of Mycobacterium tuberculosis gyrase through site-directed mutagenesis. The results show that Y577, R691 and R745 are among the key DNA-binding residues in M.tuberculosis GyrA-CTD, and that the third blade of the GyrA-CTD is the main DNA-binding region in M.tuberculosis DNA gyrase. The substitutions of Y577A, D669A, R691A, R745A and G729W led to the loss of supercoiling and relaxation activities, although they had a little effect on the drug-dependent DNA cleavage and decatenation activities, and had no effect on the ATPase activity. Taken together, these results showed that the GyrA-CTD is essential to DNA gyrase of M.tuberculosis, and promote the idea that the M.tuberculosis GyrA-CTD is a new potential target for drug design. It is the first time that the DNA-binding sites in GyrA-CTD have been identified.
由于只有II型拓扑异构酶能够引入负超螺旋，所以DNA促旋酶参与了关键的细胞过程。尽管对DNA促旋酶的其他结构域了解得更清楚，但DNA促旋酶A亚基的C末端结构域（GyrA-CTD）与DNA结合的机制尚不清楚。在这里，我们通过定点诱变研究了结核分枝杆菌促旋酶GyrA-CTD中的DNA结合位点。结果表明，Y577、R691和R745是结核分枝杆菌GyrA-CTD中关键的DNA结合残基，并且GyrA-CTD的第三个叶片是结核分枝杆菌DNA促旋酶中的主要DNA结合区域。Y577A、D669A、R691A、R745A和G729W的替换导致超螺旋和松弛活性丧失，尽管它们对药物依赖性DNA切割和解连环活性影响较小，并且对ATP酶活性没有影响。综上所述，这些结果表明GyrA-CTD对结核分枝杆菌的DNA促旋酶至关重要，并支持结核分枝杆菌GyrA-CTD是药物设计新潜在靶点的观点。这是首次鉴定出GyrA-CTD中的DNA结合位点。
