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

Academic Article
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Overview

authors

  • Deshpande, R. A.
  • Williams, G. J.
  • Limbo, O.
  • Williams, R. S.
  • Kuhnlein, J.
  • Lee, J. H.
  • Classen, S.
  • Guenther, G.
  • Russell, Paul
  • Tainer, John
  • Paull, T. T.

publication date

  • March 2014

journal

  • EMBO Journal  Journal

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.

subject areas

  • Adenosine Triphosphate
  • Cell Cycle
  • Crystallography, X-Ray
  • DNA
  • DNA Mutational Analysis
  • DNA Repair
  • DNA Repair Enzymes
  • DNA-Binding Proteins
  • Hydrolysis
  • Models, Molecular
  • Mutant Proteins
  • Protein Binding
  • Protein Conformation
  • Pyrococcus furiosus
  • Signal Transduction
  • X-Ray Diffraction
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Research

keywords

  • DNA damage signaling
  • DNA repair
  • double-strand breaks
  • protein-DNA interactions
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Identity

PubMed Central ID

  • PMC3989629

International Standard Serial Number (ISSN)

  • 0261-4189

Digital Object Identifier (DOI)

  • 10.1002/embj.201386100

PubMed ID

  • 24493214
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Additional Document Info

start page

  • 482

end page

  • 500

volume

  • 33

issue

  • 5

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