Department of Physics, Bioengineering Pohang University of Science and Technology (POSTECH), Pohang, 790-784, Korea.
Department of Chemistry, Bioengineering Pohang University of Science and Technology (POSTECH), Pohang, 790-784, Korea.
Structure. 2012 Jul 3;20(7):1264-1274. doi: 10.1016/j.str.2012.04.017. Epub 2012 Jun 7.
The mismatch repair (MMR) initiation protein MutS forms at least two types of sliding clamps on DNA: a transient mismatch searching clamp (∼1 s) and an unusually stable (∼600 s) ATP-bound clamp that recruits downstream MMR components. Remarkably, direct visualization of single MutS particles on mismatched DNA has not been reported. We have combined real-time particle tracking with fluorescence resonance energy transfer (FRET) to image MutS diffusion dynamics on DNA containing a single mismatch. We show searching MutS rotates during diffusion independent of ionic strength or flow rate, suggesting continuous contact with the DNA backbone. In contrast, ATP-bound MutS clamps that are visually and successively released from the mismatch spin freely around the DNA, and their diffusion is affected by ionic strength and flow rate. These observations show that ATP binding alters the MutS diffusion mechanics on DNA, which has a number of implications for the mechanism of MMR.
错配修复 (MMR) 起始蛋白 MutS 在 DNA 上至少形成两种类型的滑动夹：短暂的错配搜索夹（∼1 秒）和异常稳定的（∼600 秒）结合 ATP 的夹，它募集下游的 MMR 组件。值得注意的是，尚未有报道直接可视化在错配 DNA 上的单个 MutS 颗粒。我们将实时粒子追踪与荧光共振能量转移 (FRET) 相结合，以对含有单个错配的 DNA 上 MutS 的扩散动力学进行成像。我们发现，在扩散过程中，搜索 MutS 会旋转，而与离子强度或流速无关，这表明它与 DNA 骨架保持连续接触。相比之下，从错配处依次释放的结合 ATP 的 MutS 夹可以围绕 DNA 自由旋转，并且它们的扩散受离子强度和流速的影响。这些观察结果表明，ATP 结合改变了 MutS 在 DNA 上的扩散力学，这对 MMR 的机制有多种影响。
