Li Y, Carty M P, Oakley G G, Seidman M M, Medvedovic M, Dixon K
Department of Environmental Health, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA.
Environ Mol Mutagen. 2001;37(2):128-40. doi: 10.1002/em.1020.
Ataxia telangiectasia (A-T) is a human genetic disorder characterized by progressive cerebellar degeneration, hypersensitivity to ionizing radiation (IR), immunodeficiency, and high cancer risk. At the cellular level, IR sensitivity and increased frequency of spontaneous and IR-induced chromosomal breakage and rearrangements are the hallmarks of A-T. The ATM gene, mutated in this syndrome, has been cloned and codes for a protein sharing homology with DNA-PKcs, a protein kinase involved in DNA double-strand break (DSB) repair and DNA damage responses. The characteristics of the A-T cellular phenotypes and ATM gene suggest that ATM may play a role similar to that of DNA-PKcs in DSB repair and that there is a primary DNA repair defect in A-T cells. In the current study, the function of ATM in DNA DSB repair was evaluated in an in vitro system using two plasmids, carrying either an EcoRI-induced DSB within the lacZalpha gene or various endonuclease-induced DSB in the SupF suppressor tRNA gene. We found that the DSB repair efficiency in A-T nuclear extracts was comparable to, if not higher than, that in normal nuclear extracts. However, the repair fidelity in A-T nuclear extracts was decreased when repairing DSB with short 5' and 3' overhangs (<4 base pairs (bp)) or blunt ends, but not 5' 4-bp overhangs. Sequencing of the mutant plasmids revealed that deletions involving 1-6 nucleotide microhomologies were the major class of mutations in both A-T and normal extracts. However, the size of the deletions in plasmids from A-T nuclear extracts was larger than that from normal nuclear extracts. Expression of the ATM protein in A-T cells corrected the defect in DSB repair in A-T nuclear extracts. These results suggest that ATM plays a role in maintaining genomic stability by preventing the repair of DSB from an error-prone pathway.
共济失调毛细血管扩张症（A-T）是一种人类遗传性疾病，其特征为进行性小脑变性、对电离辐射（IR）敏感、免疫缺陷以及患癌风险高。在细胞水平上，IR敏感性以及自发和IR诱导的染色体断裂与重排频率增加是A-T的标志。在该综合征中发生突变的ATM基因已被克隆，其编码的蛋白质与DNA-PKcs具有同源性，DNA-PKcs是一种参与DNA双链断裂（DSB）修复和DNA损伤反应的蛋白激酶。A-T细胞表型和ATM基因的特征表明，ATM在DSB修复中可能发挥与DNA-PKcs类似的作用，并且A-T细胞存在原发性DNA修复缺陷。在当前研究中，使用两种质粒在体外系统中评估了ATM在DNA DSB修复中的功能，这两种质粒在lacZα基因内携带EcoRI诱导的DSB或在SupF抑制性tRNA基因中携带各种核酸内切酶诱导的DSB。我们发现，A-T细胞核提取物中的DSB修复效率即便不高于正常细胞核提取物，也与之相当。然而，当修复具有短5'和3'突出端（<4个碱基对（bp））或平端而非5' 4-bp突出端的DSB时，A-T细胞核提取物中的修复保真度降低。突变体质粒的测序显示，涉及1-6个核苷酸微同源性的缺失是A-T和正常提取物中主要的突变类型。然而，来自A-T细胞核提取物的质粒中的缺失大小大于来自正常细胞核提取物的质粒。在A-T细胞中表达ATM蛋白可纠正A-T细胞核提取物中DSB修复的缺陷。这些结果表明，ATM通过防止DSB从易出错途径进行修复，在维持基因组稳定性中发挥作用。
