DNA Double-Strand Break Repair Pathway Choice Is Directed by Distinct MRE11 Nuclease Activities

Abstract

SUMMARY MRE11 within the MRE11-RAD50-NBS1 (MRN) complex acts in DNA double-strand break repair (DSBR), detection and signaling; yet, how its endo- and exonuclease activities regulate DSB repair by non-homologous end-joining (NHEJ) versus homologous recombination (HR) remains enigmatic. Here we employed structure-based design with a focused chemical library to discover specific MRE11 endo- or exonuclease inhibitors. With these inhibitors we examined repair pathway choice at DSBs generated in G2 following radiation exposure. Whilst endo- or exonuclease inhibition impairs radiation-induced RPA chromatin binding, suggesting diminished resection, the inhibitors surprisingly direct different repair outcomes. Endonuclease inhibition promotes NHEJ in lieu of HR, whilst exonuclease inhibition confers a repair defect. Collectively, the results describe nuclease-specific MRE11 inhibitors, define distinct nuclease roles in DSB repair, and support a mechanism whereby MRE11 endonuclease initiates resection, thereby licensing HR followed by MRE11 exo and EXO1/BLM bidirectional resection towards and away from the DNA end, which commits to HR.

53BP1, BRCA1, and the Choice between Recombination and End Joining at DNA Double-Strand Breaks

“…A plausible model for Spo11 removal involves endonucleolytic incision of the DNA adjacent to the end by the MRX complex, followed by bidirectional exonucleolytic digestion from the nick, with MRX acting 3= to 5= (toward the break end) and Exo1 or Dna2/BLM performing long-range 5=-to-3= resection (96). The use of small-molecule inhibitors of MRN endo-and exonuclease activities has provided support for a similar model at mitotic DSBs, where Spo11 is absent but Ku is likely to be bound (97). Whether or not the human MRN complex can overcome resection inhibition by Ku in a manner analogous to Spo11 is being addressed by different groups (98,99).…”

Dna2 initiates resection at clean DNA double-strand breaks

“…In this step, the endonuclease activity of the Mre11 subunit in MRN/MRX is believed to be responsible for the cleavage, although CtIP/Sae2 has also been suggested to contain endonuclease activity (33,34,41–45). In the subsequent stage of resection, the 3′ end at the cleavage site generated by initiation is then processed by MRN and Exd2 in the 3′-5′ direction (35,46,47), whereas the 5′ end is further resected by Exo1 (together with PCNA or the 9-1-1 complex) and Dna2 (together with BLM/WRN, RPA and Cdc24) in the 5′-3′ direction (33,34,48–64). The resulting long ssDNA overhangs then promote the activation of HR and the ATR checkpoint (10,11,28,29).…”

Structure of the catalytic domain of Mre11 fromChaetomium thermophilum

“…In eukaryotes, the Mre11-Rad50-Nbs1 (MRN) complex plays a key role in the early steps of DSB repair, and its function in the initial detection and processing of DNA ends is important for the choice between resection-dependent (HR, MMEJ) and resection-independent (NHEJ) pathways (Lisby et al, 2004;Truong et al, 2013;Chiruvella et al, 2013;Shibata et al, 2014). MRN consists of a dimer of Mre11, two Rad50s and, in eukaryotes, Nbs1 (Lammens et al, 2011;Schiller et al, 2012;Mö ckel et al, 2012;Lim et al, 2011;Arthur et al, 2004;Das et al, 2010;Limbo et al, 2012).…”