ATP-driven Rad50 conformations regulate DNA tethering,end resection,and ATM checkpoint signaling |
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| Authors: | 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 |
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| Institution: | 1. The 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. Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA;3. The Scripps Research Institute, La Jolla, CA, USA;4. Department 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. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA;6. The Skaggs Institute for Chemical Biology, La Jolla, CA, USA |
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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. |
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| Keywords: | DNA damage signaling DNA repair double-strand breaks protein-DNA interactions |
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