Abstract：
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.
摘要：
在酿酒酵母中（S.酿酒酵母)，Mre11-Rad50-Xrs2（MRX）-Sae2核酸酶活性是切除具有二级结构或蛋白质块的DNA断裂所必需的，而在人类中,需要与CtIP的MRE11-RAD50-NBS1(MRN)同源物启动所有断裂的DNA末端切除.磷酸化Sae2/CtIP刺激MRX/N的核酸内切酶活性。对Mre11核酸酶激活的结构见解仅适用于缺乏Sae2/CtIP的生物体，所以对Sae2/CtIP如何激活核酸酶集合知之甚少。这里,我们使用生化和遗传分析的AlphaFold2结构模型组合揭示了Sae2激活Mre11的机制。我们显示Sae2使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建立了Sae2如何在体外和体内激活MRX，支持结构模型。最后,我们的研究揭示了人类CtIP，尽管序列差异很大，采用类似的机制来激活MRN。
