Sae2 and Rif2 regulate MRX endonuclease activity at DNA double-strand breaks in opposite manners

The Mre11-Rad50-Xrs2 (MRX) complex detects and processes DNA double-strand breaks (DSBs). Its DNA binding and processing activities are regulated by transitions between an ATP-bound state and a post-hydrolysis cutting state that is nucleolytically active. Mre11 endonuclease activity is stimulated by Sae2, whose lack increases MRX persistence at DSBs and checkpoint activation. Here we show that the Rif2 protein inhibits Mre11 endonuclease activity and is responsible for the increased MRX retention at DSBs in sae2Δ cells. We identify a Rad50 residue that is important for Rad50-Rif2 interaction and Rif2 inhibition of Mre11 nuclease. This residue is located near a Rad50 surface that binds Sae2 and is important in stabilizing the Mre11-Rad50 (MR) interaction in the cutting state. We propose that Sae2 stimulates Mre11 endonuclease activity by stabilizing a post-hydrolysis MR conformation that is competent for DNA cleavage, whereas Rif2 antagonizes this Sae2 function and stabilizes an endonuclease inactive MR conformation. © 2021 The Author(s) Phosphorylated Sae2 interacts with Rad50 and stimulates Mre11 endonuclease activity through an unknown mechanism. Marsella et al. show that Sae2 binding to the Rad50-Mre11 interface stabilizes a post-hydrolysis, endonuclease-active Mre11-Rad50 conformation. Rif2 interferes with the adoption of this conformation and inhibits Mre11 endonuclease activity.