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ATP‐driven Rad50 conformations regulate DNA tethering, end resection, and ATM checkpoint signaling

Rajashree A Deshpande, Gareth J Williams, Oliver Limbo, R Scott Williams, Jeff Kuhnlein, Ji‐Hoon Lee, Scott Classen, Grant Guenther, Paul Russell, John A Tainer, Tanya T Paull

Author Affiliations

  1. Rajashree A Deshpande1,,
  2. Gareth J Williams2,,
  3. Oliver Limbo3,
  4. R Scott Williams4,
  5. Jeff Kuhnlein1,
  6. Ji‐Hoon Lee1,
  7. Scott Classen5,
  8. Grant Guenther2,
  9. Paul Russell3,
  10. John A Tainer*,2,3,6 and
  11. Tanya T Paull*,1
  1. 1The Department of Molecular Genetics and Microbiology, The Howard Hughes Medical Institute Institute for Cellular and Molecular Biology The University of Texas at Austin, Austin, TX, USA
  2. 2Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA
  3. 3The Scripps Research Institute, La Jolla, CA, USA
  4. 4Department of Health and Human Services, Laboratory of Structural Biology, National Institute of Environmental Health Sciences US National Institutes of Health, Research Triangle Park, NC, USA
  5. 5Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA
  6. 6The Skaggs Institute for Chemical Biology, La Jolla, CA, USA
  1. * Corresponding author. Tel: 512 232 7802; Fax: 512 471 3730; E‐mail: tpaull{at}mail.utexas.edu

    Corresponding author. Tel: 510 495 2404; Fax: 510 486 6880; E‐mail: jat{at}scripps.edu

  1. These authors contributed equally to this work.

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Abstract

The Mre11‐Rad50 complex is highly conserved, yet the mechanisms by which Rad50 ATP‐driven states regulate the sensing, processing and signaling of DNA double‐strand breaks are largely unknown. Here we design structure‐based mutations in Pyrococcus furiosus Rad50 to alter protein core plasticity and residues undergoing ATP‐driven movements within the catalytic domains. With this strategy we identify Rad50 separation‐of‐function mutants that either promote or destabilize the ATP‐bound state. Crystal structures, X‐ray scattering, biochemical assays, and functional analyses of mutant PfRad50 complexes show that the ATP‐induced ‘closed’ conformation promotes DNA end binding and end tethering, while hydrolysis‐induced opening is essential for DNA resection. Reducing the stability of the ATP‐bound state impairs DNA repair and Tel1 (ATM) checkpoint signaling in Schizosaccharomyces pombe, double‐strand break resection in Saccharomyces cerevisiae, and ATM activation by human Mre11‐Rad50‐Nbs1 in vitro, supporting the generality of the P. furiosus Rad50 structure‐based mutational analyses. These collective results suggest that ATP‐dependent Rad50 conformations switch the Mre11‐Rad50 complex between DNA tethering, ATM signaling, and 5′ strand resection, revealing molecular mechanisms regulating responses to DNA double‐strand breaks.

Synopsis

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Comprehensive structural and functional studies with Rad50 separation‐of‐function mutants show how ATP binding and hydrolysis toggle the Mre11‐Rad50‐Nbs1 (MRN) complex between closed conformations promoting DNA end tethering, and open states mediating end resection.

  • Structure‐based mutations in Rad50 alter protein core plasticity and stability of the ATP‐bound state.

  • The ATP‐bound ‘closed’ state of MR promotes DNA binding and end tethering.

  • ATP hydrolysis‐induced opening of the MR complex is necessary for resection nuclease activity.

  • A balance between the ‘closed’ and ‘open’ states is essential for DNA repair and ATM checkpoint activation.

EMBO Journal (2014) 33, 482–500

  • Received June 26, 2013.
  • Revision received November 25, 2013.
  • Accepted November 28, 2013.
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