1. ATP-driven Rad50 conformations regulate DNA tethering, end resection, and ATM checkpoint signaling.
- Author
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Deshpande, Rajashree A, Williams, Gareth J, Limbo, Oliver, Williams, R Scott, Kuhnlein, Jeff, Lee, Ji‐Hoon, Classen, Scott, Guenther, Grant, Russell, Paul, Tainer, John A, and Paull, Tanya T
- Subjects
ADENOSINE triphosphate ,GENETIC regulation ,SURGICAL excision ,DNA repair ,CELLULAR signal transduction ,DNA damage ,DNA-protein interactions - 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. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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