ANALYSIS OF MUTATIONS CAUSED BY DNA DOUBLE-STRAND BREAKS PRODUCED BY A RESTRICTION ENZYME IN SHUTTLE VECTOR PLASMIDS PROPAGATED IN ATAXIA-TELANGIECTASIA CELLS

Rejoining of DNA double-strand breaks (DSB) produced by a restriction endonuclease AvaI in the supF gene in a plasmid pZ189Ava, and mutation s presumably due to the altered rejoinings were analyzed. After allowi ng the rejoining and replication of the plasmids in human cells origin ating from normal subjects and ataxia telangiectasia (AT) patients, th e plasmids were retrieved and those containing mutated supF were scree ned in an indicator strain of Escherichia coli. The proportion of corr ectly rejoined plasmids was significantly lower in AT cells than in no rmal cells, suggesting that AT cells have lower fidelity in rejoining DSB. DNA sequencing of the mutated supF genes revealed that all mutati ons were deletions or insertions occurring exactly or closely at the r ejoining site in both normal and AT cells. In AT cells, the majority o f mutations were deletions, while deletions and insertions were evenly formed in normal cells. AT cells may be deficient in the mechanism to protect the broken ends of DNA strands from the exonucleolytic digest ion.