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Structure of Mre11–Nbs1 complex yields insights into ataxia-telangiectasia–like disease mutations and DNA damage signaling

Abstract

The Mre11–Rad50–Nbs1 (MRN) complex tethers, processes and signals DNA double-strand breaks, promoting genomic stability. To understand the functional architecture of MRN, we determined the crystal structures of the Schizosaccharomyces pombe Mre11 dimeric catalytic domain alone and in complex with a fragment of Nbs1. Two Nbs1 subunits stretch around the outside of the nuclease domains of Mre11, with one subunit additionally bridging and locking the Mre11 dimer via a highly conserved asymmetrical binding motif. Our results show that Mre11 forms a flexible dimer and suggest that Nbs1 not only is a checkpoint adaptor but also functionally influences Mre11-Rad50. Clinical mutations in Mre11 are located along the Nbs1-interaction sites and weaken the Mre11-Nbs1 interaction. However, they differentially affect DNA repair and telomere maintenance in Saccharomyces cerevisiae, potentially providing insight into their different human disease pathologies.

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Figure 1: Structure of S. pombe apo Mre11cd and comparison with homologous Mre11 structures from Pyrococcus furiosus and Thermotoga maritima.
Figure 2: Structure of Nbs1mir–Mre11cd complex.
Figure 3: Structural basis for ATLD and NBS-like disease mutations.
Figure 4: Conformational impact of Nbs1 binding on Mre11 dimer configuration.
Figure 5: In vivo characterization of Mre11 latching loop–compromising mutations in S. cerevisiae.
Figure 6: Models for general architecture of eukaryotic MRN and MRN-dependent DNA DSB signaling.

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Acknowledgements

We are grateful to J. Petrini (Memorial Sloan-Kettering Cancer Center, New York) for his gift of antibodies to Mre11, Rad50 and Xrs2; members of the Hopfner lab for technical support and discussions; and M. Bennett, A. Rojowska, A. Kopetzki and C. Jung for help with experimentation. We thank the Max Planck Institute crystallization facility for crystallization trials, the staffs of the synchrotron beamlines for help with data collection and processing and SLS and ESRF for beamtime allowance. Research in the K.-P.H. lab was funded by grants from the German Research Council (SFBs 684, 646 and TR5), the German Excellence Initiative, European Commission (IP DNA repair), and US National Institutes of Health (U19AI83025). Research in the K.S. lab was funded by grants from the German Research Council (SFB 646) and the European Research Council (ERC; ERC Starting Grant, project 204522). Research in the S.P.J. lab is supported by grants from Cancer Research UK (C6/A11226), the European Research Council, the European Community's Seventh Framework Program (FP7/2007-2013) under grant agreement HEALTH-F2-2010-259893 and by core infrastructure funding from Cancer Research UK and the Wellcome Trust. S.P.J. receives his salary from the University of Cambridge, supplemented by Cancer Research UK.

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C.B.S., I.G., B.C., H.F. and C.M. designed experiments; C.B.S., F.S. and A.S. cloned constructs and purified proteins; C.B.S. and F.S. crystallized proteins; C.B.S. and K.L. determined crystal structures; I.G., C.B.S., B.C. and H.F. carried out S. cerevisiae assays; C.B.S. did analytical size exclusion experiments; C.M. carried out nuclease activity assays; K.-P.H. and C.B.S. wrote the manuscript; K.L., I.G. and B.C. contributed to the writing and S.P.J., H.F. and C.M. revised the manuscript; K.-P.H., S.P.J. and K.S. supervised the research; K.-P.H. initiated the project and designed the research.

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Correspondence to Karl-Peter Hopfner.

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Schiller, C., Lammens, K., Guerini, I. et al. Structure of Mre11–Nbs1 complex yields insights into ataxia-telangiectasia–like disease mutations and DNA damage signaling. Nat Struct Mol Biol 19, 693–700 (2012). https://doi.org/10.1038/nsmb.2323

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