Structurally distinct Mre11 domains mediate MRX functions in resection, end-tethering and DNA damage resistance

Abstract

Sae2 cooperates with the Mre11–Rad50-Xrs2 (MRX) complex to initiate resection of DNA double-strand breaks (DSBs) and to maintain the DSB ends in close proximity to allow their repair. How these diverse MRX-Sae2 functions contribute to DNA damage resistance is not known. Here, we describe mre11 alleles that suppress the hypersensitivity of sae2Δ cells to genotoxic agents. By assessing the impact of these mutations at the cellular and structural levels, we found that all the mre11 alleles that restore sae2Δ resistance to both camptothecin and phleomycin affect the Mre11 N-terminus and suppress the resection defect of sae2Δ cells by lowering MRX and Tel1 association to DSBs. As a consequence, the diminished Tel1 persistence potentiates Sgs1-Dna2 resection activity by decreasing Rad9 association to DSBs. By contrast, the mre11 mutations restoring sae2Δ resistance only to phleomycin are located in Mre11 C-terminus and bypass Sae2 function in end-tethering but not in DSB resection, possibly by destabilizing the Mre11–Rad50 open conformation. These findings unmask the existence of structurally distinct Mre11 domains that support resistance to genotoxic agents by mediating different processes.

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

“…Here, we show that the presence of the Mre11-R10T mutant variant, which bypasses Sae2 function in DNA damage resistance, causes a more efficient DSB end resection compared to wild-type Mre11. Unlike previously reported Mre11 mutant variants that suppress the DNA damage hypersensitivity and/or the resection defects of sae2Δ cells by potentiating the Sgs1-Dna2 resection activity through a lowering of MRX association with DSBs (Chen et al, 2015;Puddu et al, 2015;Cassani et al, 2018), the enhanced DSB resection in the presence of Mre11-R10T depends mainly on Exo1.…”

“…Analysis of a structural alignment of the different structures available for Mre11 orthologs (Cassani et al , ) revealed that the positions corresponding to the R10 residue were highly conserved (Fig A).…”

“…MD simulations were performed as described in Cassani et al , , by using gromacs 5.0.4 software package (http://www.gromacs.org) and the AMBER99 force field (Amadei et al , ). During the simulations, the interactions between magnesium ions (Mg 2+ ) and their coordinating residues were treated using non‐bonded model with van der Waals and electrostatic terms present in the AMBER99 force field.…”

“…Analysis of a structural alignment of the different structures available for Mre11 orthologs (Cassani et al, 2018) revealed that the positions corresponding to the R10 residue were highly conserved ( Fig 5A). A model for S. cerevisiae Mre11 (ScMre11) was obtained by homology modeling based on the structure of the Schizosaccharomyces pombe ortholog (see Materials and Methods for details).…”

“…In particular, Rad9 association with DSBs is promoted by the checkpoint kinase Tel1 (Gobbini et al , ), whose loading to DSBs requires the MRX complex (Nakada et al , ; Falck et al , ; You et al , ). As a consequence, mre11 alleles that cause a reduction in MRX binding to DSBs restore DNA damage resistance and resection in sae2∆ cells by diminishing Tel1 and Rad9 association at DSBs and therefore by relieving Sgs1‐Dna2 inhibition (Chen et al , ; Puddu et al , ; Cassani et al , ). Regarding Ku, this complex binds dsDNA ends with a much higher affinity than it does ssDNA (Mimori & Hardin, ; Griffith et al , ; Blier et al , ; Dynan & Yoo, ; Walker et al , ), suggesting that the MRX‐Sae2 endonuclease activity can create a DNA substrate that is less favorable for Ku engagement (Mimitou & Symington, ; Langerak et al , ; Chanut et al , ).…”