Identification of 2-deoxyribonolactone at the site of neocarzinostatin-induced cytosine release in the sequence d(AGC)

Neocarzinostatin- (NCS) induced release of cytosine from the deoxycytidylate residues of d(AGC) sequences of duplex oligonucleotides leaves a damaged sugar residue with intact phosphodiester linkages [Kappen, L.S., Chen, C., & Goldberg, I.H. (1988) Biochemistry 27, 4331-4340]. In order to isolate and characterize the sugar damage product, drug-treated duplex d(AGCGAGC*G) (the single target C* residue has 3H in its 5- and 5'-positions) was enzymatically digested to mononucleosides. High-pressure liquid chromatographic analysis of the digest revealed drug-induced products which could be cleanly separated by thin-layer chromatography (TLC) into two components: product a (Rf0.47) and product 1 (Rf0.87). The more polar product a was further purified by adsorption onto DEAE-Sephadex A-25. After elution with HCl and lyophilization, this material behaved like product 1 on TLC. Readjustment to alkaline pH caused its quantitative conversion back to product a. On electrophoresis product 1 behaved like a neutral compound, and the negatively charged product a migrated just behind formate. On the basis of the various chemical and biochemical characteristics of the lesion and apparent 3H abstraction by NCS from the C-1' position, it appears that the two interconvertible products a and 1 are respectively the acid (carboxylate) and lactone forms of 2-deoxyribonic acid. The structure of the sugar damage product was confirmed by gas chromatography/mass spectrometry. The amount of 2-deoxyribonolactone recovered is about 60% of the cytosine released on a molar basis, showing that it is the major, if not the only, product associated with cytosine release.(ABSTRACT TRUNCATED AT 250 WORDS)