ARTICLE Human RAD50 Deficiency in a Nijmegen Breakage Syndrome-like Disorder Regina Waltes, 1,2,9 Reinhard Kalb, 3,9 Magtouf Gatei, 4 Amanda W. Kijas, 4 Markus Stumm, 5 Alexandra Sobeck, 3 Britta Wieland, 1,2 Raymonda Varon, 6 Yaniv Lerenthal, 7 Martin F. Lavin, 4,8 Detlev Schindler, 3, * and Thilo Do ¨rk 1, * The MRE11/RAD50/NBN (MRN) complex plays a key role in recognizing and signaling DNA double-strand breaks (DSBs). Hypomorphic mutations in NBN (previously known as NBS1) and MRE11A give rise to the autosomal-recessive diseases Nijmegen breakage syndrome (NBS) and ataxia-telangiectasia-like disorder (ATLD), respectively. To date, no disease due to RAD50 deficiency has been described. Here, we report on a patient previously diagnosed as probably having NBS, with microcephaly, mental retardation, ‘bird-like’ face, and short stature. At variance with this diagnosis, she never had severe infections, had normal immunoglobulin levels, and did not develop lymphoid malignancy up to age 23 years. We found that she is compound heterozygous for mutations in the RAD50 gene that give rise to low levels of unstable RAD50 protein. Cells from the patient were characterized by chromosomal instability; radiosensitivity; failure to form DNA damage-induced MRN foci; and impaired radiation-induced activation of and downstream signaling through the ATM protein, which is defective in the human genetic disorder ataxia-telangiectasia. These cells were also impaired in G1/S cell- cycle-checkpoint activation and displayed radioresistant DNA synthesis and G2-phase accumulation. The defective cellular phenotype was rescued by wild-type RAD50. In conclusion, we have identified and characterized a patient with a RAD50 deficiency that results in a clinical phenotype that can be classified as an NBS-like disorder (NBSLD). Introduction Deficiencies in DNA double-strand break (DSB) signaling and repair are hallmarks of distinctive neurodegenerative syndromes. 1,2 Germ-line mutations in the ATM and MRE11A genes underlie the autosomal-recessive cerebellar ataxia syndromes ataxia-telangiectasia (A-T [MIM 208900]) 3 and ataxia-telangiectasia-like disorder (ATLD [MIM 604391]), 4 whereas germ-line mutations in the NBN (previ- ously termed NBS1) gene are responsible for the Nijmegen breakage syndrome (NBS [MIM 251260]), an autosomal- recessive disorder characterized by microcephaly, growth retardation, immunodeficiency, radiosensitivity, and cancer predisposition. 5,6 The MRE11/RAD50/NBN (MRN) complex is a highly conserved protein complex implicated in both homologous recombination repair (HRR) and nonhomologous end joining (NHEJ) of DNA DSBs, 7,8 in telomere maintenance, and in DNA replication. 9,10 This protein complex acts as a sensor of DSBs and recruits the ataxia telangiectasia mutated (ATM) protein to sites of the breaks, where it is activated. 11–13 The MRN complex is rapidly relocalized to nuclear foci in response to irradiation of cells. 14 It was shown that ATM, which phosphorylates NBN for activation of the S-phase checkpoint, was not required for association of the MRN complex with sites of DNA damage. 15 The complex also binds tightly to chromatin during normal S phase. 16 The initial event in recognizing and responding to DSBs is the binding of a RAD50/MRE11 heterotetramer (R/M complex) to DNA, tethering broken ends of DSBs. 17 This binding is achieved through the two DNA-binding motifs of MRE11 and is arranged as a globular domain with RAD50 Walker A and B motifs (ATPase domains). The bridging of DNA molecules is achieved through CXXC sequences in the middle part of RAD50. 18 These sequences occur at the ends of a 960-aa-long heptad repeat coiled-coil region and appear to dimerize by the coordination of a Zn 2þ ion. 19 The dynamic architecture of the MRN complex is altered for parallel orientation of the coiled coils of RAD50, favoring intercomplex association. 20 The exonuclease and endonuclease activities of MRE11 are stimulated upon asso- ciation with RAD50. 21,22 NBN also stimulates its endonu- clease activity. 23 The complete complex can partly unwind or dissociate a short DNA duplex with a 3 0 overhang, and this activity is stimulated by ATP. 23 MRN is required for full activation of ATM at the DSB. 24 As part of its engagement, ATM is recruited to DNA flanking the DSB, where it is activated by autophosphorylation. 25,26 Conversion of an inactive dimer to an active monomer is part of this process. The likely sequence of events has recently been elucidated at defined endogenous DSBs. 27,28 The initial activation of ATM appears to occur by relaxation of chromatin, followed by local disruption of the nucleo- some structure, recruitment of ATM to the MRN complex 1 Clinics of Obstetrics and Gynaecology, Hannover Medical School, D-30625 Hannover, Germany; 2 Institute of Radiation Oncology, Hannover Medical School, D-30625 Hannover, Germany; 3 Department of Human Genetics, Biozentrum, University of Wu ¨rzburg, D-97074 Wu ¨ rzburg, Germany; 4 Queensland Institute of Medical Research, Royal Brisbane Hospital, Herston, Queensland 4029, Australia; 5 Institute of Human Genetics, Otto von Guericke University, D-39120 Magdeburg, Germany; 6 Institute of Human Genetics, Alexander von Humboldt University, D-13353 Berlin, Germany; 7 Department of Human Genetics and Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, 69978 Ramat Aviv, Israel; 8 University of Queensland, Centre for Clinical Research, Royal Brisbane Hospital, Brisbane, Queensland 4029, Australia 9 These authors contributed equally to this work *Correspondence: schindler@biozentrum.uni-wuerzburg.de (D.S.), doerk.thilo@mh-hannover.de (T.D.) DOI 10.1016/j.ajhg.2009.04.010. ª2009 by The American Society of Human Genetics. All rights reserved. The American Journal of Human Genetics 84, 605–616, May 15, 2009 605