Deoxyribonucleic acid damage by neocarzinostatin chromophore: strand breaks generated by selective oxidation of C-5' of deoxyribose.
Among the lesions induced in DNA by neocarzinostatin chromophore are spontaneous and alkali-dependent base release, sugar damage, and single-strand breaks with phosphate (PO4) at their 3' ends and PO4 or nucleoside 5'-aldehyde at the 5' ends. By measuring alkali-dependent thymine release and decomposition of the 5'-terminal thymidine 5'-aldehyde in drug-cut DNA, we show that the kinetics are the same for each process and that the nucleoside aldehyde is the source of about 85% of alkali-dependent thymine release. Reduction of the 5'-aldehyde ends to 5'-hydroxyls followed by incorporation of 32P from [gamma-32P]ATP by polynucleotide kinase permits their selective quantitation. Nucleoside 5'-aldehyde so measured accounts for over 80% of the drug-generated 5' ends; the remainder have PO4 termini. Since these techniques also include the contribution of alkali-labile sites in the measurement of PO4 ends, DNA sequencing was used to measure the ends directly. Using 3'-32P end-labeled DNA restriction fragments as substrates for the drug, it was found that drug attack at a T results in mainly two bands--the stronger one represents oligonucleotide with 5'-terminal nucleoside 5'-aldehyde and may account for over 90% of a particular break. Its structure was verified by its isolation from the sequencing gel, followed by various chemical and enzymatic treatments. In each case, the mobility of the product on the gel was altered in a predictable manner. In addition to spontaneous breaks, neocarzinostatin also causes alkali-labile breaks preferentially at T residues. These sites are heterogeneous in their sensitivity to alkali and are protected by reduction.
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D001483:Base Sequence; D004256:DNA Polymerase I / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects*; D004279:DNA, Viral / Q000235:genetics; D003855:Deoxyribose / Q000378:metabolism; D053281:Enediynes; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D010957:Plasmids; D009353:Zinostatin / Q000031:analogs & derivatives / Q000494:pharmacology*
D000903:Antibiotics, Antineoplastic; D004279:DNA, Viral; D053281:Enediynes; D003855:Deoxyribose; C032372:neocarzinostatin chromophore; D009353:Zinostatin; D004256:DNA Polymerase I