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

Abstract

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 catalytic stimulation of Mus81-Eme1 depends entirely on direct phosphorylation of Eme1 by Rad3(ATR) and that while Eme1 also undergoes Chk1-mediated phosphorylation, this is not essential for catalytic modulation. Both Rad3(ATR)- and Chk1-mediated phosphorylation of Eme1 as well as the SIMs are independently critical for cell fitness in absence of Rqh1(BLM) and abrogating bimodal phosphorylation of Eme1 along with mutating the SIMs is incompatible with rqh1∆ cell viability. Our findings unravel an elaborate regulatory network that is essential for Mus81-Eme1 to fulfill functions that are essential in absence of Rqh1(BLM).