Mechanism of mismatch recognition by MutS in linear and circular DNA
Mechanism of mismatch recognition by MutS in linear and circular DNA
复制标题
MutS 线性和环状 DNA 错配识别机制
DOI：
10.1016/j.bpj.2023.11.1447
复制
发表时间：
2024
影响因子：
3.4
通讯作者：
Ansari, Anjum
中科院分区：
文献类型：
--
作者：
Baral, Saroj;Zvoda, Viktoriya;Pigli, Ying Z.;Rice, Phoebe A.;Antony, Edwin;Ansari, Anjum
复制后DNA错配修复由MutS蛋白启动，其识别单个错配/插入-缺失错误。许多研究表明，MutS感觉改变了不匹配的局部灵活性。然而，并不是所有的错配都被同样有效地识别，这仍然是令人困惑的。我们使用放置在错配附近的荧光核苷酸类似物6-MI比较了有效（T-凸起和GT）和无效（TT和TC）修复错配的内在DNA柔性/动力学（Li et al.等（2019），Int. J. Mol. Sci. 20：4271-4298）。61 bp的DNA的荧光寿命研究显示，TT和TC，从匹配的（AT）对应物难以区分的可访问的构象范围很窄。相比之下，T-凸起在两种不同的构象之间切换，而GT似乎部分未堆叠。通过激光T跳跃，我们在低特异性TC和非特异性AT上测量了几乎相同的毫秒动力学，而T凸起表现出更快的弛豫动力学（T跳跃的分辨率< 10 μs）。我们的研究验证了最近的MD模拟结果，该结果显示T凸起DNA在15 μs MD时间尺度上的快速波动（Jayaraj et al.（2023），Biophys. J. 122：3031-3043）。这些快速波动的位点可能有助于阻止扩散MutS以促进询问，并代表更容易被扭结以形成识别复合物的错配。大多数错配识别的研究都集中在扭转松弛的线性DNA寡聚体上。然而，DNA结构和变形性受到DNA拓扑结构（成环和超螺旋）的强烈影响，这反过来又会影响错配识别。我们通过将T凸起并入126 bp的DNA微环来研究DNA成环的效果。在小环的T-凸起网站单独的荧光寿命分布类似的MutS结合的复合物，和MutS的结合亲和力的小环增加了近10倍相比，线性DNA。我们的研究结果表明，弯曲应变变形的T-凸起网站的构象更容易识别MutS。
Post-replication DNA mismatch repair is initiated by MutS protein, which recognizes single mismatches/insertion-deletion errors. Many studies suggest that MutS senses altered local flexibility at the mismatch. However, all mismatches are not recognized equally efficiently, which remains puzzling. We compared intrinsic DNA flexibility/dynamics for efficiently—(T-bulge and GT) and inefficiently—(TT and TC) repaired mismatches using fluorescent nucleotide analog 6-MI placed adjacent to the mismatch (Li et al.(2019), Int. J. Mol. Sci. 20: 4271-4298). Fluorescence lifetime studies on 61-bp DNA revealed a narrow range of accessible conformations for TT and TC, indistinguishable from the matched (AT) counterpart. In contrast, T-bulge toggled between two distinct conformations while GT appeared partially unstacked. With laser T-jump, we measured nearly identical millisecond dynamics on low-specificity TC and nonspecific AT, while T-bulge exhibited much faster relaxation kinetics (< 10-μs resolution of our T-jump). Our studies validate recent MD simulations results that showed rapid fluctuations in T-bulge DNA on the 15-μs MD timescales (Jayaraj et al.(2023), Biophys. J. 122: 3031-3043). These rapidly fluctuating sites likely help stall a diffusing MutS to facilitate interrogation and represent mismatches more amenable to being kinked to form the recognition complex. Most studies of mismatch recognition have focused on torsionally relaxed, linear DNA oligomers. However, DNA structure and deformability are strongly influenced by DNA topology (looping and supercoiling), which in turn should impact mismatch recognition. We examined the effect of DNA looping by incorporating a T-bulge into a 126-bp DNA minicircle. The fluorescence lifetime distribution at the T-bulge site for minicircle alone resembled that of the MutS-bound complex, and MutS binding affinity for the minicircle increased nearly 10-fold compared with linear DNA. Our results imply that the bending strain deforms the T-bulge site to a conformation more readily recognized by MutS.