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

Antonio Marsella,Elisa Gobbini,Corinne Cassani,Renata Tisi,Elda Cannavo,Giordano Reginato,Petr Cejka,Maria Pia Longhese

doi:10.1016/j.celrep.2021.108906

Antonio Marsella, Elisa Gobbini + Show 6 more

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https://doi.org/10.1016/j.celrep.2021.108906

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Abstract

SummaryThe 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.

Highlights

DNA double-strand breaks (DSBs) are highly cytotoxic lesions that can be repaired by non-homologous end joining (NHEJ) or homologous recombination (HR) (Mehta and Haber, 2014)
NHEJ involves the direct ligation of the DSB ends, HR uses the DNA information stored in a homologous duplex DNA as a template for repair
Because Rif2 has been shown to stimulate Rad50 ATPase activity (Cassani et al, 2016; Hailemariam et al, 2019), to better understand the interplay between Sae2 and Rif2 in MRX regulation, we analyzed the effect of deleting RIF2 from sae2D cells. sae2D rif2D double-mutant cells were more resistant to camptothecin (CPT) and methyl methanesulfonate (MMS) compared with sae2D cells (Figure 1A), indicating that the lack of Rif2 partially suppresses the DNA damage sensitivity of sae2D cells

Summary

Introduction

DNA double-strand breaks (DSBs) are highly cytotoxic lesions that can be repaired by non-homologous end joining (NHEJ) or homologous recombination (HR) (Mehta and Haber, 2014). NHEJ involves the direct ligation of the DSB ends, HR uses the DNA information stored in a homologous duplex DNA as a template for repair. The obligate step of HR is the degradation of the 50-terminated DNA strands at both DSB ends to generate 30 end single-stranded DNA (ssDNA) overhangs that catalyze homologous pairing and strand invasion (Ranjha et al, 2018). Apical checkpoint proteins include Mec and Tel kinases (ATR and ATM in mammals, respectively). Once activated by damaged DNA, Tel and Mec transduce the checkpoint signals to the downstream effector kinases Rad and Chk (CHK2 and CHK1 in mammals, respectively), whose activation requires the conserved protein Rad (53BP1 in mammals) (Gilbert et al, 2001; Sweeney et al, 2005; Schwartz et al, 2002)

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