3'-Formyl phosphate-ended DNA: high-energy intermediate in antibiotic-induced DNA sugar damage.

Abstract

Under anaerobic conditions where the nitroaromatic radiation-sensitizer misonidazole substitutes for dioxygen, DNA strand breakage (gaps with phosphate residues at each end) by the nonprotein chromophore of the antitumor antibiotic neocarzinostatin (NCS-Chrom) is associated with the generation of a reactive form of formate from the C-5' of deoxyribose of thymidylate residues. Such lesions account for a minority (10-15%) of the strand breakage found in the aerobic reaction without misonidazole. Amino-containing nucleophiles such as tris(hydroxymethyl)aminomethane (Tris) and hydroxylamine act as acceptors for the activated formate. The amount of [3H formyl hydroxamate produced from DNA labeled with [5'-3H]thymidine is comparable to the spontaneously released thymine. During the course of the reaction, misonidazole undergoes a DNA-dependent reduction and subsequent conjugation with glutathione used to activate NCSChrom. From these and earlier results, we propose a possible mechanism in which the carbon-centered radical formed at C-5' by hydrogen atom abstraction by thiol-activated NCSChrom reacts anaerobically with misonidazole to form a nitroxyl-radical-adduct intermediate, which fragments to produce an oxy radical at C-5'. j3-Fragmentation results in cleavage between C-5' and C-4' with the generation of 3'-formyl phosphate-ended DNA, a high-energy form of formate, which spontaneously hydrolyzes, releasing formate and creating a 3'-phosphate end, or transfers the formyl moiety to available nucleophiles. A similar mechanism, involving dioxygen addition, is probably responsible for the 10-15% DNA gap formation in the aerobic reaction.as to position the reactive ring system containing the two acetylenic bonds close to the C-5' of deoxyribose of mainly thymidylate residues.Upon activation by thiol [glutathione in the cell (2)], the DNA-bound drug is converted into a species, presumably free-radical form, that abstracts a hydrogen atom from C-5' to generate a carbon-centered radical, whether or not dioxygen is present (3). Addition of dioxygen to the radical at C-5' forms a peroxyl radical species that undergoes reduction by thiol to generate a DNA strand break with a nucleoside 5'-aldehyde group at the 5' end and a phosphate at the 3' end.Such lesions account for more than 80% of the strand breaks produced (4). The remaining 10-15% of the strand breaks are actually gaps with phosphates at both ends. The mechanism for their production has not been clear, although it has been thought to be related to the formation of formate from C-5' (5). In addition to single-strand breaks, DNA bases (mainly thymine) are also released (6, 7), in part generating abasic sites. When nitroaromatic radiation sensitizers, such as misonidazole, are substituted for dioxygen in the reaction, gaps with phosphates at both ends constitute the main form of DNA breakage (8), and this is associated with base (mainly thymine) release and a 4-to 5-fold increase (over the aerobic reaction) in the release of a soluble material labeled with tri...

Ultraviolet light-induced cleavage of DNA in presence of iodoHoechst 33258: the sequence specificity of the reaction

“…This type of data is very similar to that obtained for neocarzinostatin (22)(23)(24) and calicheamicin 'yI (25). The detailed experiments by Goldberg and co-workers with neocarcinostatin revealed that the reaction mechanism involves production of a free-radical which abstracts a hydrogen atom from the deoxyribose 5 '-carbon, resulting in strand cleavage leaving a 3'-phosphoryl terminus and a carbonyl group at the 5' carbon.…”

Influence of thiol structure on neocarzinostatin activation and expression of DNA damage

“…At the C4'-position, breakdown of the peroxyradical partitions to form a 4'-hydroxylated abasic site (Saito et al, 1989;Frank et al, 1991;Kappen et al, 1991) or to produce a strand break with a 3'-phosphoglycolate residue and a 5'-phosphate Kappen et al, 1991). Similarly, the strand break resulting from the degradation of the C5'-peroxyradical involves either a nucleoside 5'-aldehyde at the 5'-end and a 3I-phosphate (Kappen et al, 1982;Kappen & Goldberg, 1983) or a labile 3'-formylphosphate-ended DNA fragment and a 5'-phosphate (Kappen & Goldberg, 1984;Chin et al, 1987) (Scheme I). Abstraction of the C1'-hydrogen atom, the third …”

N-formylation of lysine in histone proteins as a secondary modification arising from oxidative DNA damage

“…2). The labile phosphate ester of the formylphosphate promotes transfer of the formyl group to primary and secondary amines (20). That the 5Ј-position of deoxyribose in DNA is the most solvent accessible site in the B-helix suggests that 3Ј-formylphosphate residues and other 5Ј-oxidation products will be among the most common features of DNA strand breaks in cells (21).…”