27. King, R. W., Deshaies, R. J., Peters, J.-M. & Kirschner, M. W. How proteolysis drives the cell cycle. Science 274, 1652–1658 (1996). 28. Lorca, T. et al. Fizzy is required for activation of the APC/cyclosome in Xenopus egg extracts. EMBO J. 17, 3565–3575 (1998). 29. Rudner, A. & Murray, A. Phosphorylation by Cdc28 activates the Cdc20-dependent activity of the anaphase-promoting complex. J. Cell Biol. 149, 1377–1390 (2000). 30. Oehlen, L. J. W. M., McKinney, J. D. & Cross, F. R. Ste12 and Mcm1 regulatecellcycle dependent transcription of FAR1. Mol. Cell. Biol. 16, 2830–2837 (1996). Supplementary Information accompanies the paper on Nature’s website (http://www.nature.com/nature). Acknowledgements We thank A. Amon, K. Nasmyth, M. Tyers, E. Schwob, W. Seufert and M. Shirayama for reagents and A. Amon, K. Nasmyth, J. Roberts and M. Tyers for useful discussions and critical comments on the manuscript. We also thank C. Li and A. Doty for technical assistance and J. Schmoranzer for help with some micrographs. This work was supported bya grant from Deutsche Krebshilfe to R.W. and a PHS grant to F.C. Competing interests statement The authors declare that they have no competing financial interests. Correspondence and requests for materials should be addressed to F.C. (e-mail: fcross@rockefeller.edu). .............................................................. The Rad50 zinc-hook is a structure joining Mre11 complexes in DNA recombination and repair Karl-Peter Hopfner*†, Lisa Craig*, Gabriel Moncalian*, Robert A. Zinkel‡, Takehiko Usui§, Barbara A. L. Owenk, Annette Karcher†, Brendan Henderson{, Jean-Luc Bodmer*, Cynthia T. McMurrayk, James P. Carney{, John H. J. Petrini§ & John A. Tainer*# * Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA † Gene Center and Institute of Biochemistry, University of Munich, 81377 Munich, Germany ‡ Laboratory of Genetics, University of Wisconsin, Madison, Wisconsin 53706, USA § Molecular Biology, Memorial Sloan-Kettering CancerCenter, New York, New York 10021, USA k Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA { The Radiation Oncology Research Laboratory, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA # Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA ............................................................................................................................................................................. The Mre11 complex (Mre11–Rad50–Nbs1) is central to chromo- somal maintenance and functions in homologous recombina- tion, telomere maintenance and sister chromatid association 1–7 . These functions all imply that the linked binding of two DNA substrates occurs, although the molecular basis for this process remains unknown. Here we present a 2.2 A ˚ crystal structure of the Rad50 coiled-coil region that reveals an unexpected dimer interface at the apex of the coiled coils in which pairs of conserved Cys-X-X-Cys motifs form interlocking hooks that bind one Zn 21 ion. Biochemical, X-ray and electron microscopy data indicate that these hooks can join oppositely protruding Rad50 coiled-coil domains to form a flexible bridge of up to 1,200 A ˚ . This suggests a function for the long insertion in the Rad50 ABC-ATPase domain 8 . The Rad50 hook is functional, because mutations in this motif confer radiation sensitivity in yeast and disrupt binding at the distant Mre11 nuclease interface. These data support an architectural role for the Rad50 coiled coils in forming metal-mediated bridging complexes between two DNA-binding heads. The resulting assemblies have appropriate lengths and conformational properties to link sister chromatids in homologous recombination and DNA ends in non-homolo- gous end-joining. Orthologues of the DNA double-strand break repair nuclease Mre11 and the ATPase Rad50 exist in all kingdoms of life and function as a heterotetrameric (Mre11) 2 /(Rad50) 2 complex (M2R2) 8 . In eukaryotes, the Mre11 complex can contain a third Figure 1 Rad50 domains, sequence conservation of the CXXC motif and Zn 2þ -mediated dimerization of Rad50. a, Domain structure of Rad50 and multiple sequence alignment of the central portion of the Rad50 coiled coil showing the conserved CXXC motif. The Walker A (A) and Walker B (B) motifs are labelled in the ABC-ATPase domains (green), and the Mre11-binding site (M) and the hook construct (Hk) are labelled in the coiled-coil (CC) regions (orange). Residue numbers delineating the domains and motifs are shown for human (above) and for P. furiosus Rad50 (below). Alignment of the region encompassing the CXXC motif is conserved from bacteria to humans (dark grey, invariant cysteine residues; light grey, conserved residues). pf, P. furiosus; h, human; ce, Caenorhabditis elegans; at, Arabidopsis thaliana; sc, S. cerevisiae; sp, Schizosaccharomyces pombe; ec, E. coli; T4, bacteriophage T4. b, Representative scan of absorbance versus radius and the residuals (inset) for the equilibrium analysis of pfRad50-CXXC-L in the presence of Zn 2þ . c, d, Sedimentation equilibrium analysis (c) and sedimentation velocity analysis (d) of pfRad50-CXXC-L in the presence or absence of Zn 2þ . Zn 2þ concentration was determined by atomic absorption; obs M is the observed M (K) at 17,000 or 24,000 r.p.m.; S 20,w is the sedimentation coefficient (10 213 s); D 20,w is the diffusion constant (cm 2 s 21 ). The theoretical M deduced from the sequence is 16,628 for the monomer (M) and 33,256 for the dimer (D). letters to nature NATURE | VOL 418 | 1 AUGUST 2002 | www.nature.com/nature 562 © 2002 Nature Publishing Group