IDENTIFICATION OF DEFECTIVE ILLEGITIMATE RECOMBINATIONAL REPAIR OF OXIDATIVELY-INDUCED DNA DOUBLE-STRAND BREAKS IN ATAXIA-TELANGIECTASIA CELLS

Ataxia-telangiectasia (A-T) is an autosomal-recessive lethal human dis ease. Homozygotes suffer from a number of neurological disorders, as w ell as very high cancer incidence. Heterozygotes may also have a highe r than normal risk of cancer, particularly for the breast. The gene re sponsible for the disease (ATM) has been cloned, but its role in mecha nisms of the disease remain unknown. Cellular A-T phenotypes, such as radiosensitivity and genomic instability, suggest that a deficiency in the repair of DNA double-strand breaks (DSBs) may be the primary defe ct; however, overall levels of DSB rejoining appear normal. We used th e shuttle vector, pZ189, containing an oxidatively-induced DSB, to com pare the integrity of DSB rejoining in one normal and two A-T fibrobla st cell lines. Mutation frequencies were two-fold higher in A-T cells, and the mutational spectrum was different. The majority of the mutati ons found in all three cell lines were deletions (44-63%). The DNA seq uence analysis indicated that 17 of the 17 plasmids with deletion muta tions in normal cells occurred between short direct-repeat sequences ( removing one of the repeats plus the intervening sequences), implicati ng illegitimate recombination in DSB rejoining. The combined data from both A-T cell lines showed that 21 of 24 deletions did not involve di rect-repeats sequences, implicating a defect in the illegitimate recom bination pathway. These findings suggest that the A-T gene product may either directly participate in illegitimate recombination or modulate the pathway. Regardless, this defect is likely to be important to a m echanistic understanding of this lethal disease. (C) 1997 Elsevier Sci ence B.V.