Evidence for the role of DNA strand passage in the mechanism of action of microcin B17 on DNA gyrase
Evidence for the role of DNA strand passage in the mechanism of action of microcin B17 on DNA gyrase
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DOI：
10.1021/bi0478751
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发表时间：
2005-03-22
期刊：
影响因子：
2.9
通讯作者：
Maxwell, A
中科院分区：
文献类型：
--
作者：
Pierrat, OA;Maxwell, A
Microcin B 17（MccB 17）是一种DNA促旋酶毒素;在以前的工作中，发现这种细菌毒素缓慢且不完全地抑制由促旋酶引起的DNA超螺旋和松弛反应，并稳定切割复合物，这取决于ATP和DNA拓扑结构的存在。我们现在表明，MccB 17对促旋酶ATP酶反应和切割复合物形成的作用需要超过150个碱基对的线性DNA片段。MccB 17不能通过稳定短线性DNA片段（70个碱基对或更少）和促旋酶之间的弱相互作用来刺激ATP酶反应，与喹诺酮环丙沙星相反。然而，MccB 17可以通过缺乏其DNA包装或ATP酶结构域的促旋酶影响DNA的ATP依赖性松弛。从这些发现中，我们提出了MccB 17的作用模式，需要足够长的DNA分子，以允许通过酶的DNA门运输一个片段。此外，我们认为，MccB 17可能会捕获一个短暂的中间状态的促旋酶反应只存在于DNA链通道和酶营业额。来自胰蛋白酶处理的完整酶的MccB 17的蛋白水解特征需要DNA和ATP，并且显示出对促旋酶的C-末端47-kDa结构域的保护，表明该结构域参与毒素作用模式，并且与其在DNA链通过机制中的拟议作用一致。我们推测MccB 17的结合位点位于GyrB的C端结构域。
Microcin B 17 (MccB17) is a DNA gyrase poison; in previous work, this bacterial toxin was found to slowly and incompletely inhibit the reactions of supercoiling and relaxation of DNA by gyrase and to stabilize the cleavage complex, depending on the presence of ATP and the DNA topology. We now show that the action of MccB17 on the gyrase ATPase reaction and cleavage complex formation requires a linear DNA fragment of more than 150 base pairs. MccB17 is unable to stimulate the ATPase reaction by stabilizing the weak interactions between short linear DNA fragments (70 base pairs or less) and gyrase, in contrast with the quinolone ciprofloxacin. However, MccB17 can affect the ATP-dependent relaxation of DNA by gyrase lacking its DNA-wrapping or ATPase domains. From these findings, we propose a mode of action of MccB17 requiring a DNA molecule long enough to allow the transport of a segment through the DNA gate of the enzyme. Furthermore, we suggest that MccB17 may trap a transient intermediate state of the gyrase reaction present only during DNA strand passage and enzyme turnover. The proteolytic signature of MccB17 from trypsin treatment of the full enzyme requires DNA and ATP and shows a protection of the C-terminal 47-kDa domain of gyrase, indicating the involvement of this domain in the toxin mode of action and consistent with its proposed role in the mechanism of DNA strand passage. We suggest that the binding site of MccB17 is in the C-terminal domain of GyrB.