Institute for Condensed Matter Physics, Darmstadt University of Technology, Darmstadt, Germany.
Radiation Biology and DNA Repair, Darmstadt University of Technology, Darmstadt, Germany.
Biophys J. 2019 Apr 23;116(8):1406-1419. doi: 10.1016/j.bpj.2019.03.001. Epub 2019 Mar 19.
The repair of DNA double-strand breaks by homologous recombination is of crucial importance for maintaining genomic stability. Two major players during this repair pathway are Rad51 and Rad54. Previously, it was shown that Rad54 exists as a monomer or oligomer when bound to DNA and drives the displacement of Rad51 by translocating along the DNA. Moreover, phosphorylation of Rad54 was reported to stimulate this clearance of Rad51 from DNA. However, it is currently unclear how phosphorylation of Rad54 modulates its molecular-structural function and how it affects the activity of monomeric or oligomeric Rad54 during the removal of Rad51. To examine the impact of Rad54 phosphorylation on a molecular-structural level, we applied molecular dynamics simulations of Rad54 monomers and hexamers in the absence or presence of DNA. Our results suggest that 1) phosphorylation of Rad54 stabilizes the monomeric form by reducing the interlobe movement of Rad54 monomers and might therefore facilitate multimer formation around DNA and 2) phosphorylation of Rad54 in a higher-order hexamer reduces its binding strength to DNA, which is a requirement for efficient mobility on DNA. To further address the relationship between the mobility of Rad54 and its phosphorylation state, we performed fluorescence recovery after photobleaching experiments in living cells, which expressed different versions of the Rad54 protein. Here, we could measure that the phosphomimetic version of Rad54 was highly mobile on DNA, whereas a nonphosphorylatable mutant displayed a mobility defect. Taken together, these data show that the phosphorylation of Rad54 is a critical event in balancing the DNA binding strength and mobility of Rad54 and might therefore provide optimal conditions for DNA translocation and subsequent removal of Rad51 during homologous recombination repair.
同源重组修复 DNA 双链断裂对于维持基因组稳定性至关重要。在这个修复途径中，两个主要参与者是 Rad51 和 Rad54。以前的研究表明，Rad54 与 DNA 结合时以单体或寡聚体的形式存在，并通过沿着 DNA 易位来驱动 Rad51 的置换。此外，已有报道称 Rad54 的磷酸化可刺激 Rad51 从 DNA 上的清除。然而，目前尚不清楚 Rad54 的磷酸化如何调节其分子结构功能，以及它如何影响单体或寡聚 Rad54 在 Rad51 去除过程中的活性。为了在分子结构水平上研究 Rad54 磷酸化的影响，我们应用了 Rad54 单体和六聚体在无 DNA 或有 DNA 存在情况下的分子动力学模拟。我们的结果表明：1）Rad54 的磷酸化通过减少 Rad54 单体的叶间运动来稳定单体形式，从而可能促进 DNA 周围的多聚体形成；2）更高阶六聚体中 Rad54 的磷酸化降低了其与 DNA 的结合强度，这是在 DNA 上有效移动的要求。为了进一步探讨 Rad54 的运动性与其磷酸化状态之间的关系，我们在表达不同版本 Rad54 蛋白的活细胞中进行了光漂白后荧光恢复实验。在这里，我们可以测量到 Rad54 的磷酸模拟版本在 DNA 上具有很高的流动性，而不可磷酸化的突变体则表现出流动性缺陷。综上所述，这些数据表明 Rad54 的磷酸化是平衡 Rad54 的 DNA 结合强度和流动性的关键事件，因此可能为同源重组修复过程中的 DNA 易位和随后的 Rad51 去除提供最佳条件。
