195 RADIATION RESEARCH 158, 195–201 (2002) 0033-7587/ 02 $5.00 q 2002 by Radiation Research Society. All rights of reproduction in any form reserved. Detection of ATM Gene Mutation in Human Glioma Cell Line M059J by a Rapid Frameshift/Stop Codon Assay in Yeast Rika Tsuchida, a,b,1 Takayuki Yamada, a,1 Masatoshi Takagi, a Akira Shimada, c Chikashi Ishioka, c Yoko Katsuki, a Takashi Igarashi, b Luciana Chessa, d Domenico Delia, e Hirobumi Teraoka f and Shuki Mizutani a,2 a Department of Pediatrics and Developmental Biology, Postgraduate Medical School, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan; b Department of Pediatrics, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113- 8655, Japan; c Department of Clinical Oncology, Institute of Development, Aging and Cancer, Tohoku University, 4-1, Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan; d Department of Experimental Medicine and Pathology, University La Sapienza, Viale del Policlinico 155, 00161 Rome, Italy; e Department of Experimental Oncology, Istituto Nazionale Tumori, Via G. Venezian 1, 20133 Milan, Italy; and f Department of Pathological Biochemistry, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10, Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan Tsuchida, R., Yamada, T., Takagi, M., Shimada, A., Ishio- ka, Katsuki, Y., C., Igarashi, T., Chessa, L., Delia, D., Ter- aoka, H. and Mizutani, S. Detection of ATM Gene Mutation in Human Glioma Cell Line M059J by a Rapid Frameshift/ Stop Codon Assay in Yeast. Radiat. Res. 158, 195–201 (2002). A yeast-based frameshift/stop codon assay for examining ATM (ataxia telangiectasia mutated) mutations was estab- lished. Each of six fragments of a PCR-amplified coding se- quence for ATM is inserted in frame by homologous recom- bination into a yeast URA3 fusion protein gene, and the trans- formants are assayed for growth in the absence of uracil. The usefulness of this assay was verified in a panel of cell lines derived from individuals with homozygous and heterozygous ATM mutations. The assay was also shown to distinguish be- tween specimens with wild-type alleles and those with trun- cating mutations: a frameshift mutation or an inserted stop codon. Using this assay M059J cells, which fail to express the catalytic subunit of DNA-dependent protein kinase (PRKDC, also known as DNA-PKcs) and are hypersensitive to ionizing radiation, were found to express two different aberrant ATM transcripts: one characterized by 4776 del 133, which corre- sponds to the deletion of exon 33, and the other by 4909 ins 116. Subsequent analysis of the intron sequences revealed that 4909 ins 116 is comprised of a nucleotide sequence corre- sponding to 84013–84128 in intron 33 with a cryptic splice site. Thus the radiosensitive phenotype of M059J cells appears to be due to a defect in PRKDC and a truncating ATM mutation. q 2002 by Radiation Research Society INTRODUCTION Ataxia telangiectasia (AT) is a pleiotropic autosomal re- cessive disorder with a progressive cerebellar ataxia, bulbar 1 These authors contributed equally to this work. 2 Author to whom correspondence should be addressed at Department of Pediatrics and Developmental Biology, Postgraduate Medical School, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku,To- kyo 11 3-8519, Japan; e-mail: smizutani.ped@tmd.ac.jp. telangiectasia, and immunological deficiencies. AT patients also manifest an increased incidence of malignancies, such as malignant lymphoma, leukemia and solid tumors (1, 2). The ATM protein has been shown to play multiple key roles in intracellular signaling for cell cycle control and DNA repair/recombination in response to DNA-damaging agents (3). In response to DNA double-strand breaks (DSBs), ATM leads to the phosphorylation of several key molecules in an intricate network of proteins. Examples of such substrates are the TP53 (4, 5), MDM2 (6), CHK2 (now known as CHEK2) (7 ), BRCA1 (8), ABL (9, 10) and NBS1 proteins (11–13). The frequency of AT is estimated to be about one in 300,000 births, and AT heterozygote frequency reportedly falls between 0.68% and 7.7%, with 2.8% being a likely estimate (14–16). AT patients, and possibly AT carriers, have an increased risk of cancer, especially breast cancer (1, 16, 17 ). It was reported that not only cells from AT patients but also cells from carriers of ATM mutations have biological abnormalities, as indicated by assays of clono- genic cell survival and studies of the activation of cell cycle checkpoints in response to DSBs (18, 19), that are largely due to insufficient levels of ATM protein (20–23) or to the effect of a dominant-negative mutant ATM protein (24, 25). Since most of the mutations of the ATM gene in AT patients are truncating mutations (26, 27 ), many ATM mutations in AT carriers may also be truncations. To set up a screening test for the detection of truncating ATM mutations, a yeast- based frameshift/stop codon assay was established. We de- scribe here a method for the detection of ATM truncating mutations with Saccharomyces cerevisiae. Each of six frag- ments of a PCR-amplified coding sequence for ATM is in- serted in frame by homologous recombination into a yeast URA3 fusion protein gene, and transformants are assayed for growth in the absence of uracil. The specificity of trans- lational initiation of the URA3 gene leads to minimal en- zymatic activity in transformants harboring a frameshift