Visualizing the assembly and disassembly mechanisms of the MuB transposition targeting complex
Visualizing the assembly and disassembly mechanisms of the MuB transposition targeting complex
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DOI：
10.1074/jbc.m311883200
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发表时间：
2004-04-16
影响因子：
4.8
通讯作者：
Mizuuchi, K
中科院分区：
文献类型：
--
作者：
Greene, EC;Mizuuchi, K
MuB是噬菌体Mu复制DNA转座所必需的蛋白质，是一种ATP酶，在DNA上组装成聚合复合物。我们使用全内反射荧光显微镜观察MuB聚合物在单分子DNA上的行为。我们证明了聚合物组装是由随机成核事件引发的。成核后，聚合物组装通过涉及MuB小单元的顺序结合的机制发生。MuB与DNA的A/T富集区结合，组装成大的聚合物复合物。与此相反，MuB的A/T-丰富的区域外绑定未能组装成大的寡聚复合物。我们的数据还表明，MuB不催化多轮ATP水解，同时保持与DNA结合。相反，单个ATP被水解，然后MuB从DNA中解离。最后，我们表明，“加帽”的增强型绿色荧光蛋白-MuB聚合物与未标记的MuB大大减缓，但不停止，解离结束。这表明MuB解离通过末端依赖性机制和较慢的机制发生，其中亚基从聚合物内部解离。
MuB, a protein essential for replicative DNA transposition by the bacteriophage Mu, is an ATPase that assembles into a polymeric complex on DNA. We used total internal reflection fluorescence microscopy to observe the behavior of MuB polymers on single molecules of DNA. We demonstrate that polymer assembly is initiated by a stochastic nucleation event. After nucleation, polymer assembly occurs by a mechanism involving the sequential binding of small units of MuB. MuB that bound to A/T-rich regions of the DNA assembled into large polymeric complexes. In contrast, MuB that bound outside of the A/T-rich regions failed to assemble into large oligomeric complexes. Our data also show that MuB does not catalyze multiple rounds of ATP hydrolysis while remaining bound to DNA. Rather, a single ATP is hydrolyzed, then MuB dissociates from the DNA. Finally, we show that "capping" of the enhanced green fluorescent protein-MuB polymer ends with unlabeled MuB dramatically slows, but does not halt, dissociation. This suggests that MuB dissociation occurs through both an end-dependent mechanism and a slower mechanism wherein subunits dissociate from the polymer interior.