Meli Massimiliano, Sustarsic Marko, Craggs Timothy D, Kapanidis Achillefs N, Colombo Giorgio
Computational Biochemistry Group, Istituto di Chimica del Riconoscimento Molecolare, National Research Council of Italy Milano, Italy.
Clarendon Laboratory, Department of Physics, Biological Physics Research Group, University of Oxford Oxford, UK.
Front Mol Biosci. 2016 May 27;3:20. doi: 10.3389/fmolb.2016.00020. eCollection 2016.
Herein we investigate the molecular bases of DNA polymerase I conformational dynamics that underlie the replication fidelity of the enzyme. Such fidelity is determined by conformational changes that promote the rejection of incorrect nucleotides before the chemical ligation step. We report a comprehensive atomic resolution study of wild type and mutant enzymes in different bound states and starting from different crystal structures, using extensive molecular dynamics (MD) simulations that cover a total timespan of ~5 ms. The resulting trajectories are examined via a combination of novel methods of internal dynamics and energetics analysis, aimed to reveal the principal molecular determinants for the (de)stabilization of a certain conformational state. Our results show that the presence of fidelity-decreasing mutations or the binding of incorrect nucleotides in ternary complexes tend to favor transitions from closed toward open structures, passing through an ensemble of semi-closed intermediates. The latter ensemble includes the experimentally observed ajar conformation which, consistent with previous experimental observations, emerges as a molecular checkpoint for the selection of the correct nucleotide to incorporate. We discuss the implications of our results for the understanding of the relationships between the structure, dynamics, and function of DNA polymerase I at the atomistic level.
在此，我们研究了DNA聚合酶I构象动力学的分子基础，这些基础构成了该酶复制保真度的基础。这种保真度由构象变化决定，这些变化在化学连接步骤之前促进对错误核苷酸的排斥。我们报告了一项全面的原子分辨率研究，该研究涉及处于不同结合状态且起始于不同晶体结构的野生型和突变型酶，使用了总时长约为5毫秒的广泛分子动力学（MD）模拟。通过结合内部动力学和能量分析的新方法来检查所得轨迹，旨在揭示特定构象状态（去）稳定化的主要分子决定因素。我们的结果表明，保真度降低的突变的存在或三元复合物中错误核苷酸的结合倾向于促进从封闭结构向开放结构的转变，通过一系列半封闭中间体。后者包括实验观察到的微开构象，与先前的实验观察结果一致，它作为选择要掺入的正确核苷酸的分子检查点出现。我们讨论了我们的结果对于在原子水平上理解DNA聚合酶I的结构、动力学和功能之间关系的意义。
