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 double-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 oligonucleotides that have been designed to contain a specific ds-cleavage site. The BLM induced ss/ds-cleavage ratios ranged from approximately 3.3 to approximately 5.8 at 4 degrees C, with the most efficient ds-cleavage involving the thymidines of a 5'-GTAC/5'-GTAC site. Double-strand cleavage was 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-cleavage 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 oligonucleotides has greatly facilitated quantitative analysis of the ds-cleavage process (Absalon et al., 1995).
Absalon
M J
MJ
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
Kozarich
J W
JW
Stubbe
J
J
D001483:Base Sequence; D001665:Binding Sites; D001761:Bleomycin / Q000378:metabolism* / Q000494:pharmacology / Q000031:analogs & derivatives; D004247:DNA / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism*
D004277:DNA, Single-Stranded; D009841:Oligonucleotides; C030187:cobalt bleomycin; C023503:iron bleomycin; D001761:Bleomycin; D004247:DNA; D015246:Deoxyribonuclease EcoRI