The action of the bacterial toxin microcin B17 - Insight into the cleavage-religation reaction of DNA gyrase
The action of the bacterial toxin microcin B17 - Insight into the cleavage-religation reaction of DNA gyrase
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DOI：
10.1074/jbc.m304516200
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发表时间：
2003-09-12
影响因子：
4.8
通讯作者：
Maxwell, A
中科院分区：
文献类型：
--
作者：
Pierrat, OA;Maxwell, A
我们研究了细菌毒素微菌素B17（MccB 17）对大肠杆菌DNA回旋酶反应的影响。MccB 17减慢但不完全抑制DNA超螺旋和促旋酶的松弛反应。对促旋酶的裂解-再连接平衡进行动力学分析，以确定毒素对正向（裂解）和反向（再连接）反应的影响。一个简单的机制，两个连续的可逆反应与切口的DNA中间体被用来模拟切割和再连接的动力学。MccB 17对裂解和再连接动力学的作用与喹诺酮类药物环丙沙星和oxolinic acid的作用进行了比较。与松弛的DNA作为底物，只有少量的回旋酶切割复合物，观察到与MccB 17在ATP的情况下，而核苷酸的存在下显着增强的切割和再连接反应的毒素的效果。相比之下，环丙沙星，oxolinic酸，和Ca 2+显示出较低的依赖于ATP稳定的切割复合物。MccB 17通过增加第二平衡的正向速率常数（k（2））来提高DNA切割的总速率。相比之下，环丙沙星通过对两种平衡的正向和反向速率常数的组合效应来增加切割的DNA的量。基于这些结果和MccB 17只缓慢抑制超螺旋和松弛反应的观察，我们提出了MccB 17与促旋酶相互作用的模型。
We have examined the effects of the bacterial toxin microcin B17 (MccB17) on the reactions of Escherichia coli DNA gyrase. MccB17 slows down but does not completely inhibit the DNA supercoiling and relaxation reactions of gyrase. A kinetic analysis of the cleavage-religation equilibrium of gyrase was performed to determine the effect of the toxin on the forward ( cleavage) and reverse (religation) reactions. A simple mechanism of two consecutive reversible reactions with a nicked DNA intermediate was used to simulate the kinetics of cleavage and religation. The action of MccB17 on the kinetics of cleavage and religation was compared with that of the quinolones ciprofloxacin and oxolinic acid. With relaxed DNA as substrate, only a small amount of gyrase cleavage complex is observed with MccB17 in the absence of ATP, whereas the presence of the nucleotide significantly enhances the effect of the toxin on both the cleavage and religation reactions. In contrast, ciprofloxacin, oxolinic acid, and Ca2+ show lesser dependence on ATP to stabilize the cleavage complex. MccB17 enhances the overall rate of DNA cleavage by increasing the forward rate constant (k(2)) of the second equilibrium. In contrast, ciprofloxacin increases the amount of cleaved DNA by a combined effect on the forward and reverse rate constants of both equilibria. Based on these results and on the observations that MccB17 only slowly inhibits the supercoiling and relaxation reactions, we suggest a model of the interaction of MccB17 with gyrase.