Energetics of base flipping at a DNA mismatch site confined at the latch constriction of α-hemolysin.
Energetics of base flipping at a DNA mismatch site confined at the latch constriction of α-hemolysin.
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DOI：
10.1039/c6fd00058d
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发表时间：
2016-12-12
影响因子：
3.4
通讯作者：
White HS
中科院分区：
文献类型：
--
作者：
Johnson RP;Perera RT;Fleming AM;Burrows CJ;White HS
当胞嘧啶-胞嘧啶错配对被限制在 α-溶血素 (αHL) 纳米孔的 2.4 nm 闩锁收缩处时，观察到独特的两态调制电流特征。我们之前推测这种调制是由于错配位点的碱基翻转造成的。碱基翻转是一种具有重要生物学意义的机制，其中单个碱基从 DNA 螺旋堆叠中旋转 180°。这是酶能够进入碱基进行修复操作而不干扰螺旋整体结构的机制。在这里，α-HL 内单个双链 DNA 双链体的温度依赖性离子通道记录用于导出不稳定胞嘧啶-胞嘧啶错配位点处胞嘧啶碱基翻转的热力学 (ΔH、ΔS) 和动力学 (Ea) 参数。将位于 αHL 闩锁处的胞嘧啶翻转出螺旋的测量活化能 (18 ± 1 kcal mol−1) 与之前通过 NMR 测量和潜在平均力计算报告的错配位点碱基翻转的活化能相当。我们认为 αHL 纳米孔是在单分子水平上测量 dsDNA 构象变化的有用工具。
Unique, two-state modulating current signatures are observed when a cytosine-cytosine mismatch pair is confined at the 2.4 nm latch constriction of the α-hemolysin (αHL) nanopore. We have previously speculated that the modulation is due to base flipping at the mismatch site. Base flipping is a biologically significant mechanism in which a single base is rotated out of the DNA helical stack by 180°. It is the mechanism by which enzymes are able to access bases for repair operations without disturbing the global structure of the helix. Here, temperature dependent ion channel recordings of individual double-stranded DNA duplexes inside α-HL are used to derive thermodynamic (ΔH, ΔS) and kinetic (Ea) parameters for base flipping of a cytosine at an unstable cytosine-cytosine mismatch site. The measured activation energy for flipping a cytosine located at the latch of αHL out of the helix (18 ± 1 kcal mol−1) is comparable to that previously reported for base flipping at mismatch sites from NMR measurements and potential mean force calculations. We propose that the αHL nanopore is a useful tool for measuring conformational changes in dsDNA at the single molecule level.
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