SPECIFIC DETECTION OF C-4' HYDROXYLATED ABASIC SITES GENERATED BY BLEOMYCIN AND NEOCARZINOSTATIN IN DNA

A new, general method for detection and quantitation of C-4′ hydroxylated abasic sites generated by bleomycin and neocarzinostatin in DNA was described. The specific detection method was based on a chemical and enzymatic transformation of C-4′ hydroxylated abasic sites to deoxynucleoside 3′-(3-pyridazinylmethyl) phosphates. Enzymatic digestion of 3′-(3-pyridazinylmethyl) 2′-deoxycytidylyl-(3′-5′)-2′-deoxy-3′-guanylate (7) with snake venom phosphodiesterase (s.v. PDE) and alkaline phosphatase (AP) gave 2′-deoxycytidine and 3′-(3-pyridazinylmethyl) 2′-deoxyguanylate (4) in high yields, indicating that a pyridazine-substituted phosphodiester bond at the 3′-end resists digestion with s.v. PDE. Enzymatic digestion of d(CGCGAATTCGCG) treated with photoactivated green cobalt-peplomycin (Co·PEM) with s.v. PDE and AP following treatment with aqueous hydrazine was examined. Consistent with the previous results on the cleavage of this dodecanucleotide, 4 was obtained as a major product. Photoactivated Co·PEM also mediated spontaneous thymine release from poly(dA-dT) with formation of 5 and 6. Digestion of Co·PEM-treated calf thymus DNA having a C-4′ hydroxylated abasic site gave pyridazine derivatives 3-6 and the total amount of 3-6 corresponded well to the sum of spontaneously released free bases. Also investigated was the formation of 3-6 in Fe·PEM-and neocarzinostatin-(NCS-) mediated degradation of calf thymus DNA. Hydrazine treatment of their reaction mixtures followed by enzymatic digestion produced pyridazine derivatives 3-6, indicating that C-4′ hydroxylated abasic sites are actually produced in calf thymus DNA. Quantitative analysis indicated that C-4′ hydroxylation is estimated to be a minimum of 17% of the total event caused by the action of NCS on calf thymus DNA. © 1990, American Chemical Society. All rights reserved.