School of Chemistry, National University of Ireland, Galway (NUIG), Galway, Ireland.
Nucleic Acids Res. 2011 Jan;39(2):767-80. doi: 10.1093/nar/gkq812. Epub 2010 Sep 28.
Uracil (U) can be found in DNA as a mismatch paired either to adenine (A) or to guanine (G). Removal of U from DNA is performed by a class of enzymes known as uracil-DNA-glycosylases (UDG). Recent studies suggest that recognition of U-A and U-G mismatches by UDG takes place via an extra-helical mechanism. In this work, we use molecular dynamics simulations to analyze the structure, dynamics and open base pair kinetics of U-A base pairs relative to their natural T-A counterpart in 12 dodecamers. Our results show that the presence of U does not alter the local conformation of B-DNA. Breathing dynamics and base pair closing kinetics are only weakly dependent on the presence of U versus T, with open T-A and U-A pairs lifetimes in the nanosecond timescale. Additionally, we observed spontaneous base flipping in U-A pairs. We analyze the structure and dynamics for this event and compare the results to available crystallographic data of open base pair conformations. Our results are in agreement with both structural and kinetic data derived from NMR imino proton exchange measurements, providing the first detailed description at the molecular level of elusive events such as spontaneous base pair opening and flipping in mismatched U-A sequences in DNA. Based on these results, we propose that base pair flipping can occur spontaneously at room temperature via a 3-step mechanism with an open base pair intermediate. Implications for the molecular basis of U recognition by UDG are discussed.
尿嘧啶（U）可以在 DNA 中作为错配碱基与腺嘌呤（A）或鸟嘌呤（G）配对。U-DNA-糖基化酶（UDG）是一类可以将 U 从 DNA 中移除的酶。最近的研究表明，UDG 通过一种额外的螺旋机制识别 U-A 和 U-G 错配。在这项工作中，我们使用分子动力学模拟来分析 12 个十二聚体中 U-A 碱基对相对于其自然 T-A 碱基对的结构、动力学和打开碱基对动力学。我们的结果表明，U 的存在不会改变 B-DNA 的局部构象。呼吸动力学和碱基对闭合动力学仅与 U 与 T 的存在弱相关，打开的 T-A 和 U-A 碱基对的寿命在纳秒范围内。此外，我们观察到 U-A 碱基对中的自发碱基翻转。我们分析了该事件的结构和动力学，并将结果与开放碱基对构象的可用晶体学数据进行了比较。我们的结果与从 NMR 亚氨基质子交换测量中得出的结构和动力学数据一致，为在 DNA 中难以捉摸的事件（如自发碱基对打开和翻转）提供了分子水平的详细描述。基于这些结果，我们提出碱基对翻转可以通过 3 步机制在室温下自发发生，其中包含一个打开的碱基对中间体。讨论了 UDG 识别 U 的分子基础的含义。
