SEQUENCE-SPECIFIC DOUBLE-STRAND CLEAVAGE OF DNA BY FE BLEOMYCIN .1. THE DETECTION OF SEQUENCE-SPECIFIC DOUBLE-STRAND BREAKS USING HAIRPIN OLIGONUCLEOTIDES

A new method is described for the evaluation of sequence-specific doub le-strand (ds) cleavage of DNA by Fe-bleomycin (BLM). The method uses high-resolution polyacrylamide gel electrophoresis to separate single- strand (ss) and ds-cleavage products derived from hairpin oligonucleot ides that have been designed to contain a specific ds-cleavage site. T he BLM induced ss/ds-cleavage ratios ranged from similar to 3.3 to sim ilar to 5.8 at 4 degrees C, with the most efficient ds-cleavage involv ing the thymidines of a 5'-GTAC/5'-GTAC site. Double-strand cleavage w as not detected at several sites that were shown to yield significant ss-breaks. A study of the ss/ds-cleavage ratio at the 5'-GTAC/5'-GTAC site revealed that the ratio remained constant over a 70-fold range in concentration of Fe-BLM and extent of DNA degradation. The ss/ds-clea vage ratios at three sites studied were not significantly affected by the presence of ''inert'' Co(III)-BLM. The results are consistent with the proposal of Steighner and Povirk (1990) that a single molecule of Fe-BLM is responsible for ds-cleavage. The use of these hairpin oligo nucleotides has greatly facilitated quantitative analysis of the ds-cl eavage process (Absalon et al., 1995).