Molecular insights into the activation of Mre11-Rad50 endonuclease activity by Sae2/CtIP

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. [Display omitted] •AlphaFold2 reveals how phosphorylated yeast Sae2 can interact with Mre11 and Rad50•Phosphorylated Sae2 stabilizes Mre11-Rad50-Xrs2 in a conformation ready to cleave DNA•Structural models are supported by several pairs of compensatory mutations•Sae2 and CtIP may function similarly despite drastic sequence divergence Nicolas et al. unveil the structural model of yeast Sae2 in complex with Mre11 and Rad50 using AlphaFold2, supported by mutagenesis. They show how phosphorylated Sae2 can stabilize the cutting state of Mre11 and highlight structural rearrangements that may have occurred during evolution from a common ancestor of Sae2 and human CtIP.