Regulation of Mus81-Eme1 structure-specific endonuclease by Eme1 SUMO-binding and Rad3^ATR kinase is essential in the absence of Rqh1^BLM helicase

by Cédric Giaccherini, Sarah Scaglione, Stéphane Coulon, Pierre-Marie Dehé, Pierre-Henri L. Gaillard

The Mus81-Eme1 structure-specific endonuclease is crucial for the processing of DNA recombination and late replication intermediates. In fission yeast, stimulation of Mus81-Eme1 in response to DNA damage at the G2/M transition relies on Cdc2^CDK1 and DNA damage checkpoint-dependent phosphorylation of Eme1 and is critical for chromosome stability in absence of the Rqh1^BLM helicase. Here we identify Rad3^ATR checkpoint kinase consensus phosphorylation sites and two SUMO interacting motifs (SIM) within a short N-terminal domain of Eme1 that is required for cell survival in absence of Rqh1^BLM. We show that direct phosphorylation of Eme1 by Rad3^ATR is essential for catalytic stimulation of Mus81-Eme1. Chk1-mediated phosphorylation also contributes to the stimulation of Mus81-Eme1 when combined with phosphorylation of Eme1 by Rad3^ATR. Both Rad3^ATR- and Chk1-mediated phosphorylation of Eme1 as well as the SIMs are critical for cell fitness in absence of Rqh1^BLM and abrogating bimodal phosphorylation of Eme1 along with mutating the SIMs is incompatible with rqh1Δ cell viability. Our findings unravel an elaborate regulatory network that relies on the poorly structured N-terminal domain of Eme1 and which is essential for the vital functions Mus81-Eme1 fulfills in absence of Rqh1^BLM.