Mre11 Dimers Coordinate DNA End Bridging and Nuclease Processing in Double-Strand-Break Repair

Department of Molecular Biology, Scripps Research Institute, 10550 North Torrey Pines Road, MB4, La Jolla, CA 92037, USA.
Cell (Impact Factor: 32.24). 11/2008; 135(1):97-109. DOI: 10.1016/j.cell.2008.08.017
Source: PubMed

ABSTRACT Mre11 forms the core of the multifunctional Mre11-Rad50-Nbs1 (MRN) complex that detects DNA double-strand breaks (DSBs), activates the ATM checkpoint kinase, and initiates homologous recombination (HR) repair of DSBs. To define the roles of Mre11 in both DNA bridging and nucleolytic processing during initiation of DSB repair, we combined small-angle X-ray scattering (SAXS) and crystal structures of Pyrococcus furiosus Mre11 dimers bound to DNA with mutational analyses of fission yeast Mre11. The Mre11 dimer adopts a four-lobed U-shaped structure that is critical for proper MRN complex assembly and for binding and aligning DNA ends. Further, mutations blocking Mre11 endonuclease activity impair cell survival after DSB induction without compromising MRN complex assembly or Mre11-dependant recruitment of Ctp1, an HR factor, to DSBs. These results show how Mre11 dimerization and nuclease activities initiate repair of DSBs and collapsed replication forks, as well as provide a molecular foundation for understanding cancer-causing Mre11 mutations in ataxia telangiectasia-like disorder (ATLD).

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Available from: John Tainer, Sep 28, 2015
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    • "Reflecting the above requirements, cells defective in resection components display HR defects and hypersensitivity to various DNA-damaging agents. This is well illustrated by Saccharomyces cerevisiae cells harbouring defects in the Mre11–Rad50–Xrs2 (MRX) complex, which binds and juxtaposes the two ends of a DSB (Williams et al, 2008) and, through Mre11 catalytic functions, provides nuclease activities involved in DSB processing (Furuse et al, 1998; Williams et al, 2008; Stracker & Petrini, 2011). Once a clean, partially resected 5 0 end has been generated, the enzymes Exo1 and Sgs1/Dna2 are then thought to act, generating extensive ssDNA regions needed for effective HR (Mimitou & Symington, 2008; Zhu et al, 2008). "
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    ABSTRACT: DNA double-strand break (DSB) repair by homologous recombination (HR) requires 3' single-stranded DNA (ssDNA) generation by 5' DNA-end resection. During meiosis, yeast Sae2 cooperates with the nuclease Mre11 to remove covalently bound Spo11 from DSB termini, allowing resection and HR to ensue. Mitotic roles of Sae2 and Mre11 nuclease have remained enigmatic, however, since cells lacking these display modest resection defects but marked DNA damage hypersensitivities. By combining classic genetic suppressor screening with high-throughput DNA sequencing, we identify Mre11 mutations that strongly suppress DNA damage sensitivities of sae2∆ cells. By assessing the impacts of these mutations at the cellular, biochemical and structural levels, we propose that, in addition to promoting resection, a crucial role for Sae2 and Mre11 nuclease activity in mitotic DSB repair is to facilitate the removal of Mre11 from ssDNA associated with DSB ends. Thus, without Sae2 or Mre11 nuclease activity, Mre11 bound to partly processed DSBs impairs strand invasion and HR. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.
    The EMBO Journal 04/2015; 34(11). DOI:10.15252/embj.201590973 · 10.43 Impact Factor
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    • "One thing that can guide flexible enzyme-substrate recognition and allows flexibility of the enzyme's binding site is the negatively charged DNA and an appropriate positive patch on the repair enzyme surface (Huffman et al., 2005; Mol et al., 2000; Slupphaug et al., 1996). Such positive patches are seen on both Mre11 and Rad50 (Hopfner et al., 2001) and are consistent with subsequent DNA-complex crystal structures (Rojowska et al., 2014; Williams et al., 2008). Indeed electrostatics are part of DNA mimicry that can regulate DNA repair processes (Mol et al., 1995; Putnam et al., 1999; Putnam and Tainer, 2005). "
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    ABSTRACT: The Mre11-Rad50-Nbs1 (MRN) complex is a dynamic macromolecular machine that acts in the first steps of DNA double strand break repair, and each of its components has intrinsic dynamics and flexibility properties that are directly linked with their functions. As a result, deciphering the functional structural biology of the MRN complex is driving novel and integrated technologies to define the dynamic structural biology of protein machinery interacting with DNA. Rad50 promotes dramatic long-range allostery through its coiled-coil and zinc-hook domains. Its ATPase activity drives dynamic transitions between monomeric and dimeric forms that can be modulated with mutants modifying the ATPase rate to control end joining versus resection activities. The biological functions of Mre11's dual endo- and exonuclease activities in repair pathway choice were enigmatic until recently, when they were unveiled by the development of specific nuclease inhibitors. Mre11 dimer flexibility, which may be regulated in cells to control MRN function, suggests new inhibitor design strategies for cancer intervention. Nbs1 has FHA and BRCT domains to bind multiple interaction partners that further regulate MRN. One of them, CtIP, modulates the Mre11 excision activity for homologous recombination repair. Overall, these combined properties suggest novel therapeutic strategies. Furthermore, they collectively help to explain how MRN regulates DNA repair pathway choice with implications for improving the design and analysis of cancer clinical trials that employ DNA damaging agents or target the DNA damage response.
    Progress in Biophysics and Molecular Biology 01/2015; 117(2-3). DOI:10.1016/j.pbiomolbio.2014.12.004 · 2.27 Impact Factor
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    • "Mre11–Rad50 is another multidomain nuclease, which has endonuclease and exonuclease activities (Lim et al. 2011; Shibata et al. 2014). The Mre11 dimer undergoes domain rotation to facilitate the substrate cleavage (Williams et al. 2008; Sung et al. 2014). Thus, it is possible that FAN1 also exhibits processive exonuclease activity via the repeated up and down motions of the four domains. "
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    ABSTRACT: Fanconi anemia (FA) is an autosomal recessive genetic disorder caused by defects in any of 15 FA genes responsible for processing DNA interstrand cross-links (ICLs). The ultimate outcome of the FA pathway is resolution of cross-links, which requires structure-selective nucleases. FA-associated nuclease 1 (FAN1) is believed to be recruited to lesions by a monoubiquitinated FANCI–FANCD2 (ID) complex and participates in ICL repair. Here, we determined the crystal structure of Pseudomonas aeruginosa FAN1 (PaFAN1) lacking the UBZ (ubiquitin-binding zinc) domain in complex with 5′ flap DNA. All four domains of the right-hand-shaped PaFAN1 are involved in DNA recognition, with each domain playing a specific role in bending DNA at the nick. The six-helix bundle that binds the junction connects to the catalytic viral replication and repair (VRR) nuclease (VRR nuc) domain, enabling FAN1 to incise the scissile phosphate a few bases distant from the junction. The six-helix bundle also inhibits the cleavage of intact Holliday junctions. PaFAN1 shares several conserved features with other flap structure-selective nucleases despite structural differences. A clamping motion of the domains around the wedge helix, which acts as a pivot, facilitates nucleolytic cleavage. The PaFAN1 structure provides insights into how archaeal Holliday junction resolvases evolved to incise 5′ flap substrates and how FAN1 integrates with the FA complex to participate in ICL repair.
    Genes & development 09/2014; 28(28):2276-2290. DOI:10.1101/gad.248492.114 · 10.80 Impact Factor
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