NATURE | VOL 405 | 25 MAY 2000 | www.nature.com 473 letters to nature ................................................................. Functional link between ataxia-telangiectasia and Nijmegen breakage syndrome gene products Song Zhao*², Yi-Chinn Weng*², Shyng-Shiou F. Yuan*²³, Yi-Tzu Lin*², Hao-Chi Hsu*, Suh-Chin J. Lin*, Elvira Gerbino*, Mei-hua Song*, Mal  gorzata Z. Zdzienicka§, Richard A. Gattik, Jerry W. Shay¶, Yael Ziv#, Yosef Shiloh# & Eva Y.-H. P. Lee* * Department of Molecular Medicine/Institute of Biotechnology, The University of Texas Health Science Centerat San Antonio, San Antonio, Texas 78245-3207, USA § MGC-Department of Radiation Genetics and Chemical Mutagenesis, Leiden University, LUMC, Leiden, The Netherlands k Department of Pathology, University of California Los Angeles, Los Angeles, California 90095, USA ¶ Department of Cell Biology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390-9039, USA # Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel ² These authors contributed equally to this work .............................................................................................................................................. Ataxia-telangiectasia (A-T) and Nijmegen breakage syndrome (NBS) are recessive genetic disorders with susceptibility to cancer and similar cellular phenotypes 1 . The protein product of the gene responsible for A-T, designated ATM, is a member of a family of kinases characterized by a carboxy-terminal phospha- tidylinositol 3-kinase-like domain 2,3 . The NBS1 protein is speci- ®cally mutated in patients with Nijmegen breakage syndrome and forms a complex with the DNA repair proteins Rad50 and Mre11 4±7 . Here we show that phosphorylation of NBS1, induced by ionizing radiation, requires catalytically active ATM. Com- plexes containing ATM and NBS1 exist in vivo in both untreated cells and cells treated with ionizing radiation. We have identi®ed two residues of NBS1, Ser 278 and Ser 343 that are phosphorylated in vitro by ATM and whose modi®cation in vivo is essential for the cellular response to DNA damage. This response includes S-phase checkpoint activation, formation of the NBS1/Mre11/Rad50 nuclear foci and rescue of hypersensitivity to ionizing radiation. Together, these results demonstrate a biochemical link between cell-cycle checkpoints activated by DNA damage and DNA repair in two genetic diseases with overlapping phenotypes. The cellular response to DNA damage is complex and includes cell-cycle checkpoint activation, DNA repair and changes in gene transcription 8±11 . Cell lines representative of the inherited cancer- prone human diseases ataxia-telangiectasia (A-T) and Nijmegen breakage syndrome (NBS) are hypersensitive to ionizing radiation and have defects in DNA-damage-activated cell-cycle checkpoints 1 . Upon DNA damage, ATM phosphorylates p53 (refs 12, 13), and Brca1 (ref. 14). ATM is required for phosphorylation of Chk2 kinase 15 and Rad51 (refs 16, 17) induced by ionizing radiation. NBS1 is an integral component of the Mre11/Rad50/NBS1 nuclease complex 4±7 which is important in the repair of DNA double-strand breaks 18 . To examine whether a signalling cascade exists between ATM and NBS1, we studied whether NBS1 was posttranslationally modi®ed following treatment with ionizing radiation. The NBS1 monoclonal antibody speci®cally recognized a protein with a relative molecular mass of 95,000 (M r 95K) in all human cell lines examined except those established from NBS patients (data not shown). Although the electrophoretic mobility of the 95K NBS1 protein was constant throughout the cell cycle, treatment of cells with 10 Gy g-irradiation resulted in a slower migrating form of NBS1 (Fig. 1a). The mobility of the altered form of NBS1 immunoprecipitated from irradiated cells reverted to that of NBS1 from undamaged cells upon incuba- tion with phosphatase, indicating that NBS1 becomes phosphory- lated in response to DNA damage by ionizing radiation (Fig. 1b, compare lane 6 with lanes 2 and 5). Cell lines lacking functional ATM protein were used to examine whether ATM is required for phosphorylation of NBS1 after DNA damage. The ionizing radiation- or bleomycin (0.1 U per ml)- induced phosphorylation of NBS1 was not detected in these cells ³Present address: Department of Obstetrics and Gynecology, Kaohsiung Medical University, No. 100, Shih-Chuan 1st Road, Kaohsiung 80708, Taiwan. Figure 1 ATM is required for DNA damage-induced phosphorylation of NBS1. a, Mobility shift of NBS1 in response to DNA damage during the cell cycle. Synchronized T24 cells were irradiated with 10 Gy g-irradiation. Lysates (50 mg protein) of untreated cells and cells 1 h after ionizing radiation (IR) treatment were analysed by western blotting with anti- NBS1 antibody. NBS1 pp , phosphorylated form of NBS1. Mre11 is included as a protein loading control. b, Phosphorylation and mobility shift of NBS1. Lysates from untreated (lanes 1±4) and IR-treated (lanes 5±8) human lymphoblast NAT10 cells were immunoprecipitated with anti-NBS1 antibody. Immunoprecipitates were incubated with phosphatase (PPase) in the absence or presence of phosphatase inhibitor. Lysates from untreated (lane 1) and IR-treated cells (lane 8) were included as control. c, Phosphorylation of NBS1 upon DNA damage and replication block in AT22IJE-T/pEBS7 (A-T) and AT22IJE-T/pEBS7-YZ5 (A-T cells complemented with ATM). d, Kinetics of IR- induced modi®cation of NBS1. Cell lysates were prepared at the indicated time points after IR treatment. © 2000 Macmillan Magazines Ltd