ATP-driven Rad50 conformations regulate DNA tethering, end resection, and ATM checkpoint signaling.
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.
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.
Rajashree A
RA
Deshpande
Gareth J
GJ
Williams
Oliver
O
Limbo
R Scott
RS
Williams
Jeff
J
Kuhnlein
Ji-Hoon
JH
Lee
Scott
S
Classen
Grant
G
Guenther
Paul
P
Russell
John A
JA
Tainer
Tanya T
TT
Paull
D000255:Adenosine Triphosphate / Q000378:metabolism*; D002453:Cell Cycle; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006868:Hydrolysis; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D020027:Pyrococcus furiosus / Q000235:genetics / Q000254:growth & development / Q000378:metabolism* / Q000502:physiology; D015398:Signal Transduction; D014961:X-Ray Diffraction
D004268:DNA-Binding Proteins; D050505:Mutant Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D045643:DNA Repair Enzymes