Single-Molecule FRET TACKLE Reveals Highly Dynamic Mismatched DNA-MutS Complexes
Single-Molecule FRET TACKLE Reveals Highly Dynamic Mismatched DNA-MutS Complexes
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DOI：
10.1021/bi901871u
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发表时间：
2010-04-13
期刊：
影响因子：
2.9
通讯作者：
Erie, Dorothy A.
中科院分区：
文献类型：
--
作者：
Sass, Lauryn E.;Lanyi, Cherie;Erie, Dorothy A.
DNA错配修复（MMR）的第一步是MutS和MutS同源物识别DNA错配或核苷酸插入/缺失（IDL）。为了研究MutS错配复合物的构象特性，我们使用单分子荧光共振能量转移（smFRET）来研究MutS诱导的DNA在GT错配处弯曲的动力学。的FRET测量表明，MutS GT错配识别复合物是高度动态的，经历许多国家之间的构象转变与不同程度的DNA弯曲。由于数据的复杂性，我们开发了一种分析方法，称为FRET TACKLE，其中我们结合联合收割机直接分析的FRET转换与检查的动力学寿命，以确定所有的构象状态和特征的结合和构象平衡的动力学。数据显示，MutS-GT复合物可以存在于六种不同的构象中，它们的寿命相差多达20倍，并且表现出变化2个数量级的相互转化率。为了进一步深入了解GT-MutS复合物的动力学性质并加强我们分析的有效性，我们用Monte Carlo模拟补充了我们的实验数据。综上所述，我们的研究结果表明，MutS错配复合物的动力学可以控制不同DNA错配的修复效率。最后，除了揭示MutS DNA相互作用的这些重要的生物学意义外，这种FRET TACKLE方法将使其他生物系统的复杂动力学分析成为可能。
The first step in DNA mismatch repair (MMR) is the recognition of DNA mismatches or nucleotide insertions/deletions (IDLs) by MutS and MutS homologues. To investigate the conformational properties of MutS-mismatch complexes, we used single-molecule fluorescence resonance energy transfer (smFRET) to examine the dynamics of MutS-induced DNA bending at a GT mismatch. The FRET measurements reveal that the MutS GT mismatch recognition complex is highly dynamic, undergoing conformational transitions between many states with different degrees of DNA bending. Due to the complexity of the data, we developed an analysis approach, called FRET TACKLE, in which we combine direct analysis of FRET transitions with examination of kinetic lifetimes to identify all of the conformational states and characterize the kinetics of the binding and conformational equilibria. The data reveal that MutS-GT complexes can reside in six different conformations, which have lifetimes that differ by as much as 20-fold and exhibit rates of interconversion that vary by 2 orders of magnitude. To gain further insight into the dynamic properties of GT-MutS complexes and to bolster the validity of our analysis, we complemented our experimental data with Monte Carlo simulations. Taken together, our results suggest that the dynamics of the MutS mismatch complex could govern the efficiency of repair of different DNA mismatches. Finally, in addition to revealing these important biological implications of MutS DNA interactions, this FRET TACKLE method will enable the analysis of the complex dynamics of other biological systems.