Regulation of Mus81-Eme1 structure-specific endonuclease by Eme1 SUMO-binding and Rad3ATR kinase is essential in the absence of Rqh1BLM 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 Cdc2CDK1 and DNA damage checkpoint-dependent phosphorylation of Eme1 and is critical for chromosome stability in absence of the Rqh1BLM helicase. Here we identify Rad3ATR 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 Rqh1BLM. We show that direct phosphorylation of Eme1 by Rad3ATR 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 Rad3ATR. Both Rad3ATR- and Chk1-mediated phosphorylation of Eme1 as well as the SIMs are critical for cell fitness in absence of Rqh1BLM 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 Rqh1BLM.

CHK2 Regulates MUS81-EME2 Function in Response to Replication Stress and BRCA2 Deficiency

“…Since formation of DSBs at the replication fork can lead to gross chromosomal rearrangements, activity of the MUS81 endonuclease is normally blocked or negatively-regulated in S-phase 27 . In yeast, the Mus81 complex is inhibited during normal replication or following transient arrest by Cds1, while checkpoint kinases stimulate its function, cooperating with Cdks, in response to DNA damage in S-phase or, in budding yeast, when Rqh1 is absent 22,24,25,46,47 .…”

“…CC-BY-NC-ND 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is MUS81 and negatively regulates its function during S-phase in S. pombe [22][23][24][25] . In human cells, both CHK1 and CHK2 are activated during replication stress, however, CHK1 is the true functional Cds1 homologue although structurally CHK2 is the homologue of Cds1 26 .…”

“…In fission yeast, Mus81/Eme1 function is controlled by Cdc2 CDK1 and Chk1 kinases in response to DNA damage, while the checkpoint kinase Cds1 CHK2 interacts through its FHA domain with 4 MUS81 and negatively regulates its function during S-phase in S. pombe [22][23][24][25] . In human cells, both CHK1 and CHK2 are activated during replication stress, however, CHK1 is the true functional Cds1 homologue although structurally CHK2 is the homologue of Cds1 26 .…”