The DNA cleavage pathway of iron bleomycin. Strand scission precedes deoxyribose 3-phosphate bond cleavage

Abstract

DNA strand scission initiated by bleomycin is a multistep process. Three C-C or C-O bonds are broken, releasing base propenal, a nucleic base derivative with deoxyribose carbons 1-3. Either C-3'-(phosphate-O) cleavage or C-3'-C-4' plus C-1'-(ring-O) bond cleavages could cause strand cleavage. To determine the sequence of bond breakage, d(CAAGCTTG) duplex was examined for rates of 1) strand scission, monitored by the hyperchromicity of cleavage-induced denaturation; 2) base propenal formation, monitored by 1H NMR spectroscopy; 3) 5'-terminal phosphomonoester formation, monitored by 31P NMR spectroscopy. Strand scission occurred with t 1/2 = 4.1 +/- 0.5 min at 4 degrees C, faster than base propenal formation (t 1/2 = 6.7 +/- 0.3 min). Thus newly cleaved DNA includes a base propenal precursor (t 1/2 = 2-3 min). The 5'-phosphate terminus forms (t 1/2 = 7.4 +/- 0.8 min) concurrently with base propenal. Since strand scission precedes phosphomonoester formation, strand scission cannot arise from C-3'-(phosphate-O) cleavage. Instead, the base propenal precursor must be linked to the future 5'-phosphate terminus, with strand scission arising from a combination of C-3'-C-4' and C-1'-(ring-O) bond cleavages. These results provide experimental support for a recently proposed mechanism that accommodates an early oxygen attack at C-4' and 2'-deprotonation without requiring simultaneous strand scission and 5'-phosphate terminus formation.