Joining of correct and incorrect DNA double-strand break ends in normal human and ataxia telangiectasia fibroblasts

Chromosomal aberrations are believed to result from the incorrect joining o f DNA double-strand breaks (DSBs). In an attempt to investigate induction a nd rejoining quality of DSBs following ionizing radiation exposure in speci fic genomic locations of mammalian DNA, an experimental approach based on S outhern hybridization of single-copy probes to NatI restriction fragments w as developed. Induction of DSBs is measured from the decrease of the band i ntensity representing the unbroken restriction fragment. An increase in int ensity of the hybridization band following repair incubation determines rec onstitution of the original restriction fragment and thus rejoining of corr ect DNA ends. We investigated the dose dependence of DSB misrejoining using X-ray doses of 5, 10, 20, 40, and 80 Gy and provide evidence that the numb er of misrejoined DSBs exceeds, for the same doses used, the number of cyto genetically visible aberrations by an order of magnitude, reflecting the hi gher resolution of our assay. Induction of DSBs and joining of correct and incorrect break ends were further investigated in cells from a patient with the cancer-prone disease ataxia telangiectasia (AT) and in heterozygous AT cells. We found, compared to normal cells, identical induction rates and i dentical kinetics for joining correct ends following an 80-Gy X-ray exposur e. After 5 and 10 Gy, however, AT homozygotes showed a 50% elevation in the proportion of breaks that are not correctly rejoined. These data indicate a defect in the accuracy of DSB rejoining in AT cells that may account for radiation sensitivity and the occurrence of the high level of chromosomal a berrations observed in AT cells. Genes Chromosomes Cancer 27:59-68, 2000. ( C) 2000 Wiley-Liss, Inc.