Institut Curie, PSL University, Sorbonne Université, CNRS UMR3244, Dynamics of Genetic Information, 75005 Paris, France.
Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198 Gif-sur-Yvette, France.
Mol Cell. 2024 Jun 20;84(12):2223-2237.e4. doi: 10.1016/j.molcel.2024.05.019. Epub 2024 Jun 12.
In Saccharomyces cerevisiae (S. cerevisiae), Mre11-Rad50-Xrs2 (MRX)-Sae2 nuclease activity is required for the resection of DNA breaks with secondary structures or protein blocks, while in humans, the MRE11-RAD50-NBS1 (MRN) homolog with CtIP is needed to initiate DNA end resection of all breaks. Phosphorylated Sae2/CtIP stimulates the endonuclease activity of MRX/N. Structural insights into the activation of the Mre11 nuclease are available only for organisms lacking Sae2/CtIP, so little is known about how Sae2/CtIP activates the nuclease ensemble. Here, we uncover the mechanism of Mre11 activation by Sae2 using a combination of AlphaFold2 structural modeling of biochemical and genetic assays. We show that Sae2 stabilizes the Mre11 nuclease in a conformation poised to cleave substrate DNA. Several designs of compensatory mutations establish how Sae2 activates MRX in vitro and in vivo, supporting the structural model. Finally, our study uncovers how human CtIP, despite considerable sequence divergence, employs a similar mechanism to activate MRN.
在酿酒酵母（Saccharomyces cerevisiae）中，Mre11-Rad50-Xrs2（MRX）-Sae2 核酸酶活性对于具有二级结构或蛋白质块的 DNA 断裂的切除是必需的，而在人类中，需要 MRE11-RAD50-NBS1（MRN）同源物与 CtIP 来启动所有断裂的 DNA 末端切除。磷酸化的 Sae2/CtIP 可刺激 MRX/N 的内切核酸酶活性。由于缺乏 Sae2/CtIP 的生物体可获得 Mre11 核酸酶激活的结构见解，因此对于 Sae2/CtIP 如何激活核酸酶酶系知之甚少。在这里，我们使用生化和遗传测定的 AlphaFold2 结构建模组合，揭示了 Sae2 对 Mre11 激活的机制。我们表明，Sae2 将 Mre11 核酸酶稳定在准备切割底物 DNA 的构象中。几种补偿性突变的设计确定了 Sae2 如何在体外和体内激活 MRX，支持了结构模型。最后，我们的研究揭示了尽管存在相当大的序列差异，人类 CtIP 如何采用类似的机制来激活 MRN。
