The MRX complex regulates Exo1 resection activity by altering DNA end structure

Abstract

Homologous recombination is triggered by nucleolytic degradation (resection) of DNA double-strand breaks (DSBs). DSB resection requires the Mre11-Rad50-Xrs2 (MRX) complex, which promotes the activity of Exo1 nuclease through a poorly understood mechanism. Here, we describe the Mre11-R10T mutant variant that accelerates DSB resection compared to wild-type Mre11 by potentiating Exo1-mediated processing. This increased Exo1 resection activity leads to a decreased association of the Ku complex to DSBs and an enhanced DSB resection in G1, indicating that Exo1 has a direct function in preventing Ku association with DSBs. Molecular dynamics simulations show that rotation of the Mre11 capping domains is able to induce unwinding of double-strand DNA (dsDNA). The R10T substitution causes altered orientation of the Mre11 capping domain that leads to persistent melting of the dsDNA end. We propose that MRX creates a specific DNA end structure that promotes Exo1 resection activity by facilitating the persistence of this nuclease on the DSB ends, uncovering a novel MRX function in DSB resection.

RBX1 prompts degradation of EXO1 to limit the homologous recombination pathway of DNA double-strand break repair in G1 phase

“…It has been well demonstrated that EXO1 functions in the end resection and HR pathway of DSB repair in the G2 phase and is associated with its phosphorylation by CDK1/2 [17,18]. Theoretically, the function of EXO1 is stringently restrained in G1 phase, where the HR pathway does not work due to a lack of the homologous DNA template for repair.…”

Local unwinding of double-strand DNA ends by the MRX complex promotes Exo1 processing activity

“…This persistent unwinding can account for both the enhanced access to DNA ends and processing activity of Exo1 by Mre11-R10T. In fact, easily melted dsDNA ends facilitate in vitro the resection activity of Exo1 and its stimulation by MRX 9 . Furthermore, substitution with alanine of both arginine 368 and 412 residues, through which the Mre11-R10T capping domain contacts the dsDNA, reduces the ability of Mre11-R10T to promote Exo1-mediated DSB resection 9 , indicating that the increased Exo1 activity depends on the interaction between the Mre11 capping domain and the dsDNA.…”

“…Exo1 is able to degrade the 5ʹ-terminated strand within dsDNA and, therefore, it in principle does not require an helicase activity to unwind the dsDNA 1 . However, our recent characterization of the hypermorphic Saccharomyces cerevisiae Mre11-R10T mutant variant has allowed to demonstrate that the strand-separation function of MRX is important to stimulate Exo1 resection activity 9 . In particular, the mre11-R10T mutation accelerates DSB resection compared to wild type Mre11 by potentiating the processing activity of Exo1, whose association to DSBs is increased in mre11-R10T cells.…”

“…The DNA unwinding activity of MRX has been hypothesized to stem from a conformational change of the complex 10 . Molecular dynamic simulations on wild type Mre11 dimer with dsDNA show that the DNA ends rapidly interact with the two capping domains of the Mre11 dimer and such arrangements causes a strain and partial unwinding of the dsDNA molecule (Figure 1, left) 9 . The same experiment performed on Mre11-R10T dimer shows that the capping domain of one Mre11-R10T subunit undergoes an abnormal rotation that leads this domain to wedge in between the two DNA strands and to persistently melt the dsDNA ends (Figure 1, right) 9 .…”

Uncoupling Sae2 Functions in Downregulation of Tel1 and Rad53 Signaling Activities

“…The resulting nick generates an entry site for the Mre11 exonuclease, which degrades back toward the DSB end in the 39-59 direction, and for the long-range resection nucleases Exo1 and Dna2 that degrade away from the DSB in the 59-39 direction (Mimitou and Symington 2008;Zhu et al 2008;Cejka et al 2010;Niu et al 2010;Garcia et al 2011;Nimonkar et al 2011;Shibata et al 2014;Reginato et al 2017;Wang et al 2017). In addition to a nucleolytic activity in the vicinity of the broken DNA ends, MRX has a structural role in recruiting and promoting the activity of Exo1 and Dna2 at DNA DSBs (Cejka et al 2010;Nicolette et al 2010;Niu et al 2010;Shim et al 2010;Cannavo et al 2013;Gobbini et al 2018).…”