SINGLE BASE-PAIR DELETIONS INDUCED BY BLEOMYCIN AT POTENTIAL DOUBLE-STRAND CLEAVAGE SITES IN THE APRT GENE OF STATIONARY-PHASE CHINESE-HAMSTER OVARY D422 CELLS

One possible mechanism for the generation of deletion mutations is ina ccurate repair of DNA double-strand breaks. In an attempt to detect su ch aberrant repair events in intact cells, confluent stationary phase cultures of chinese hamster ovary D422 cells, which are hemizygous for aprt, were treated for two days with low concentrations of bleomycin, and aprt mutant clones were selected and analyzed by polymerase chain reaction and DNA sequencing. Bleomycin was quite mutagenic in station ary phase cells, increasing the mutant frequency by five to 40-fold at 5 to 50% survival. While spontaneous mutations generated under these conditions were predominantly base substitutions, the majority of the bleomycin-induced mutations were very small deletions, with lesser num bers of large deletions/rearrangements and base substitutions. Althoug h the small deletions tended to be clustered in several short segments of the gene, nucleosome positioning studies indicated that there was no consistent phasing of nucleosomes in aprt, suggesting that the clus tering was due to sequence specificity rather than chromatin structure . About half of the bleomycin-induced mutations were single-base-pair (-1) deletions, and the majority of these involved deletion of one C i n a G-C-n sequence (n greater than or equal to 2). At such sites, bleo mycin is known to induce double-strand breaks by fragmentation of deox yribose moieties at the same sequence position in bath strands, result ing in a blunt-ended double-strand break with 5'-phosphate and 5'-phos phoglycolate tel mini. Thus, this sequence specificity is consistent w ith a model in which bleomycin-induced -1 deletions are generated by a double-strand break rejoining process involving removal of phosphogly colate moieties from both 3' ends, followed by blunt-end ligation. The results support the view that repair of free radical-mediated double- strand breaks in mammalian cells in G(1)/G(0) phase can be effected by such simple end-joining mechanisms, without the need for homologous r ecombination.