Single-molecule views of MutS on mismatched DNA.
Single-molecule views of MutS on mismatched DNA.
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DOI：
10.1016/j.dnarep.2014.02.014
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发表时间：
2014-08
期刊：
影响因子：
3.8
通讯作者：
Fishel, Richard
中科院分区：
文献类型：
--
作者：
Lee, Jong-Bong;Cho, Won-Ki;Park, Jonghyun;Jeon, Yongmoon;Kim, Daehyung;Lee, Seung Hwan;Fishel, Richard
关键词：
在DNA复制或重组过程中发生的碱基对错配会降低遗传稳定性或反过来增加遗传多样性。错配修复（MMR）的遗传学和生物物理机制自近50年前被发现以来得到了广泛的研究。MMR是一种链特异性的切除-再合成反应，由MutS同源物（MSH）与不匹配的核苷酸结合而引发。MSH错配结合信号随后被传递到直接下游的MutL同源物（MLH/PMS） MMR组分，并最终到达开始切除的远链断裂位点。几十年来，信号传递的机制一直存在争议。我们利用单分子福斯特共振能量转移（smFRET）、荧光跟踪（smFT）和偏振全反射荧光（smP-TIRF）研究了单水热菌MutS （TaqMutS）粒子在错配DNA上的相互作用和动态行为。我们确定，Taq-MutS在与螺旋双工DNA连续接触时，通过一维（1D）热波动驱动的旋转扩散，形成了一个早期钳形，以在~ 1s间隔内寻找不匹配。当MutS遇到不匹配时，它停留约3 s以将结合的ADP交换为ATP （ADP→ATP交换）。TaqMutS结合ATP诱导了一个非常稳定的钳形（约10分钟），它从错配中滑出，并在一维热扩散的驱动下沿着相邻的双工DNA移动。atp结合的滑动夹在与DNA不连续接触时自由旋转。MSH蛋白序列的可视化表明，atp结合的滑动夹从不匹配中分离，允许多个不匹配相关的加载事件。这些直接观察结果为理解MMR过程中MSH蛋白的分子机制提供了关键线索。
Base-pair mismatches that occur during DNA replication or recombination can reduce genetic stability or conversely increase genetic diversity. The genetics and biophysical mechanism of mismatch repair (MMR) has been extensively studied since its discovery nearly 50 years ago. MMR is a strand-specific excision-resynthesis reaction that is initiated by MutS homolog (MSH) binding to the mismatched nucleotides. The MSH mismatch-binding signal is then transmitted to the immediate downstream MutL homolog (MLH/PMS) MMR components and ultimately to a distant strand scission site where excision begins. The mechanism of signal transmission has been controversial for decades. We have utilized single molecule Forster Resonance Energy Transfer (smFRET), Fluorescence Tracking (smFT) and Polarization Total Internal Reflection Fluorescence (smP-TIRF) to examine the interactions and dynamic behaviors of single Thermus aquaticus MutS (TaqMutS) particles on mismatched DNA. We determined that Taq-MutS forms an incipient clamp to search for a mismatch in ∼1 s intervals by 1-dimensional (1D) thermal fluctuation-driven rotational diffusion while in continuous contact with the helical duplex DNA. When MutS encounters a mismatch it lingers for ∼3 s to exchange bound ADP for ATP (ADP → ATP exchange). ATP binding by TaqMutS induces an extremely stable clamp conformation (∼10 min) that slides off the mismatch and moves along the adjacent duplex DNA driven simply by 1D thermal diffusion. The ATP-bound sliding clamps rotate freely while in discontinuous contact with the DNA. The visualization of a train of MSH proteins suggests that dissociation of ATP-bound sliding clamps from the mismatch permits multiple mismatch-dependent loading events. These direct observations have provided critical clues into understanding the molecular mechanism of MSH proteins during MMR.
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