Deglycosylation susceptibility and base-pairing stability of 2'-deoxyoxanosine in oligodeoxynucleotide

We have demonstrated recently that nitrous acid or nitric oxide converts 2'-deoxyguanosine (dGuo) into 2'-deoxyoxanosine (dOxo) [Suzuki, T., Yamaoka, R., Nishi, M., Ide, H., & Makino, K. (1996) J. Am. Chem. Soc. 118, 2515-2516]. In the present study, we have measured susceptibility of the N-glycosidic bond of dOxo to spontaneous hydrolysis and its base-pairing stability to evaluate the biological significance of dOxo as a new lesion in DNA. When oligodeoxynucleotide d(T5OT6) (O = dOxo), isolated from nitrous acid-treated d(T5GT6), was incubated at pH 4.0 and 70 degrees C, hydrolysis of the N-glycosidic bond of dOxo occurred with a first-order rate constant. Comparison of the rate constants with those of dGuo and dXao indicates that the N-glycosidic bond of dOxo was as stable as that of dGuo in d(T5GT6) and hydrolyzed 44-fold more slowly than that of 2'-deoxyxanthosine (dXao), a simultaneously generated damage by nitrous acid and nitric oxide. For the estimation of the base-pairing stability, UV melting curves were measured for the duplexes of d(T5OT6).d(A6NA5) (N = A, G, C, and T) at neutral pH. The Tm values obtained were 15.3, 14.1, 19.3, and 16. 3 degrees C for N = A, G, C, and T, respectively, which are much lower than that of the intact duplex containing a G.C pair at the same position [d(T5GT6).d(A6CA5), Tm = 32.8 degrees C] but comparable with those of d(T5XT6).d(A6NA5) (X = dXao, Tm = 14.8-22.3 degrees C). CD spectra of the four duplexes containing dOxo showed preservation of the structure of the intact duplex at low temperature. UV and NMR pH-titration studies indicated the pKa for the ring-opening and -closing equilibrium to be 9.4, implying that dOxo is in the ring-closed form at physiological pH. This structure appears to be not suitable geometrically for the hydrogen bond formation with a specific counter base, thus causing equally low Tm values for all the counter bases. Consequently, these results imply that dOxo, a novel DNA lesion, may have an important and unique role in mutagenic events in cells.