Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Nucleic Acids Res. 2018 Aug 21;46(14):7309-7322. doi: 10.1093/nar/gky503.
DNA polymerase β (pol β) plays a central role in the DNA base excision repair pathway and also serves as an important model polymerase. Dynamic characterization of pol β from methyl-TROSY 13C-1H multiple quantum CPMG relaxation dispersion experiments of Ile and Met sidechains and previous backbone relaxation dispersion measurements, reveals transitions in μs-ms dynamics in response to highly variable substrates. Recognition of a 1-nt-gapped DNA substrate is accompanied by significant backbone and sidechain motion in the lyase domain and the DNA binding subdomain of the polymerase domain, that may help to facilitate binding of the apoenzyme to the segments of the DNA upstream and downstream from the gap. Backbone μs-ms motion largely disappears after formation of the pol β-DNA complex, giving rise to an increase in uncoupled μs-ms sidechain motion throughout the enzyme. Formation of an abortive ternary complex using a non-hydrolyzable dNTP results in sidechain motions that fit to a single exchange process localized to the catalytic subdomain, suggesting that this motion may play a role in catalysis.
DNA 聚合酶 β（pol β）在 DNA 碱基切除修复途径中发挥核心作用，同时也是一个重要的模型聚合酶。通过对 Ile 和 Met 侧链的甲基-TROSY 13C-1H 多量子 CPMG 弛豫弥散实验和以前的 backbone 弛豫弥散测量，对 pol β 的动态特征进行了研究，结果表明，其在 μs-ms 动力学方面发生了变化，以响应高度可变的底物。识别 1-nt 缺口 DNA 底物伴随着裂解酶结构域和聚合酶结构域的 DNA 结合亚结构域中的 backbone 和侧链运动，这可能有助于促进 apoenzyme 与缺口上下游 DNA 片段的结合。形成 pol β-DNA 复合物后，backbone μs-ms 运动基本消失，导致整个酶中未偶联的 μs-ms 侧链运动增加。使用不可水解的 dNTP 形成一个无活性的三元复合物，导致侧链运动符合定位于催化亚结构域的单个交换过程，这表明这种运动可能在催化中发挥作用。
