Dissecting the roles of MuB in Mu transposition: ATP regulation of DNA binding is not essential for target delivery.
Dissecting the roles of MuB in Mu transposition: ATP regulation of DNA binding is not essential for target delivery.
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剖析 MuB 在 Mu 转座中的作用：DNA 结合的 ATP 调节对于靶标传递并不重要。
DOI：
10.1073/pnas.0805868105
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发表时间：
2008
影响因子：
11.1
通讯作者：
Baker,TaniaA
中科院分区：
文献类型：
--
作者：
Schweidenback,CaterinaTH;Baker,TaniaA
MuA转座酶和它的激活蛋白MuB之间的协作对于正确调节转座是必不可少的。MuB激活MuA催化活性，选择靶DNA，并刺激转座到选定的靶位点。选择合适的靶DNA需要MuB ATP酶的ATP水解。通过将MuB与位点特异性DNA结合蛋白Arc阻遏物融合，我们产生了一种MuB变体，该变体可以独立于ATP选择靶DNA。这种Arc-MuB融合蛋白使我们能够测试MuB的ATP结合和水解是否是刺激转座到选定DNA中所必需的，这一过程称为靶向递送。我们发现，与融合蛋白，MuB依赖性的目标交付有效地发生在ATP水解的条件下，通过突变或使用ADP被阻止。相反，在不存在核苷酸的情况下未检测到递送。这些数据表明，ATP和MuA调节的DNA结合活性的MuB是不是必不可少的目标交付，但MuB激活MuA严格需要核苷酸结合的MuB。此外，我们发现融合蛋白指导转座到其自身结合位点的40-750 bp内的DNA区域。综上所述，这些结果表明，MuB的靶向递送是MuB刺激MuA同时将MuA拴系到所选靶DNA的能力的结果。这种拴系激活物模型为蛋白质刺激控制靶位点选择的其他例子提供了一个有吸引力的解释。
Collaboration between MuA transposase and its activator protein, MuB, is essential for properly regulated transposition. MuB activates MuA catalytic activity, selects target DNA, and stimulates transposition into the selected target site. Selection of appropriate target DNA requires ATP hydrolysis by the MuB ATPase. By fusing MuB to a site-specific DNA-binding protein, the Arc repressor, we generated a MuB variant that could select target DNA independently of ATP. This Arc-MuB fusion protein allowed us to test whether ATP binding and hydrolysis by MuB are necessary for stimulation of transposition into selected DNA, a process termed target delivery. We find that with the fusion proteins, MuB-dependent target delivery occurs efficiently under conditions where ATP hydrolysis is prevented by mutation or use of ADP. In contrast, no delivery was detected in the absence of nucleotide. These data indicate that the ATP- and MuA-regulated DNA-binding activity of MuB is not essential for target delivery but that activation of MuA by MuB strictly requires nucleotide-bound MuB. Furthermore, we find that the fusion protein directs transposition to regions of the DNA within 40–750 bp of its own binding site. Taken together, these results suggest that target delivery by MuB occurs as a consequence of the ability of MuB to stimulate MuA while simultaneously tethering MuA to a selected target DNA. This tethered-activator model provides an attractive explanation for other examples of protein-stimulated control of target site selection.