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

Abstract

Chromosomal aberrations are believed to result from the incorrect joining of DNA double-strand breaks (DSBs). In an attempt to investigate induction and rejoining quality of DSBs following ionizing radiation exposure in specific genomic locations of mammalian DNA, an experimental approach based on Southern hybridization of single-copy probes to NotI restriction fragments was developed. Induction of DSBs is measured from the decrease of the band intensity representing the unbroken restriction fragment. An increase in intensity of the hybridization band following repair incubation determines reconstitution of the original restriction fragment and thus rejoining of correct 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 number of misrejoined DSBs exceeds, for the same doses used, the number of cytogenetically visible aberrations by an order of magnitude, reflecting the higher 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 identical kinetics for joining correct ends following an 80-Gy X-ray exposure. 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 aberrations observed in AT cells. Genes Chromosomes Cancer 27:59-68, 2000.