Department of Chemistry, Yeungnam University, Gyeonsan-city 38541, Republic of Korea.
School of Life Science and Technology, Tokyo Institute of Technology, 2-12-1 Ookayama, Meguro-ku, Tokyo 152-8550, Japan.
Nucleic Acids Res. 2018 Mar 16;46(5):2548-2559. doi: 10.1093/nar/gky048.
Mg2+ ion stimulates the DNA strand exchange reaction catalyzed by RecA, a key step in homologous recombination. To elucidate the molecular mechanisms underlying the role of Mg2+ and the strand exchange reaction itself, we investigated the interaction of RecA with Mg2+ and sought to determine which step of the reaction is affected. Thermal stability, intrinsic fluorescence, and native mass spectrometric analyses of RecA revealed that RecA binds at least two Mg2+ ions with KD ≈ 2 mM and 5 mM. Deletion of the C-terminal acidic tail of RecA made its thermal stability and fluorescence characteristics insensitive to Mg2+ and similar to those of full-length RecA in the presence of saturating Mg2+. These observations, together with the results of a molecular dynamics simulation, support the idea that the acidic tail hampers the strand exchange reaction by interacting with other parts of RecA, and that binding of Mg2+ to the tail prevents these interactions and releases RecA from inhibition. We observed that binding of the first Mg2+ stimulated joint molecule formation, whereas binding of the second stimulated progression of the reaction. Thus, RecA is actively involved in the strand exchange step as well as bringing the two DNAs close to each other.
镁离子刺激 RecA 催化的 DNA 链交换反应，这是同源重组的关键步骤。为了阐明镁离子和链交换反应本身作用的分子机制，我们研究了 RecA 与镁离子的相互作用，并试图确定反应的哪个步骤受到影响。RecA 的热稳定性、本征荧光和天然质谱分析表明，RecA 至少结合两个镁离子，KD≈2mM 和 5mM。RecA 的 C 端酸性尾巴缺失使其热稳定性和荧光特性对镁离子不敏感，并且在存在饱和镁离子的情况下与全长 RecA 的特性相似。这些观察结果，以及分子动力学模拟的结果，支持了这样一种观点，即酸性尾巴通过与 RecA 的其他部分相互作用阻碍链交换反应，而镁离子与尾巴的结合阻止了这些相互作用，并使 RecA 摆脱抑制。我们观察到，第一个镁离子的结合刺激了联合分子的形成，而第二个镁离子的结合则刺激了反应的进行。因此，RecA 不仅参与链交换步骤，还将两个 DNA 分子彼此拉近。
