Suzuki T, Matsumura Y, Ide H, Kanaori K, Tajima K, Makino K
Institute of Advanced Energy, Kyoto University, Gokanosho, Uji 611, Japan.
Biochemistry. 1997 Jul 1;36(26):8013-9. doi: 10.1021/bi970166l.
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.
我们最近已证明，亚硝酸或一氧化氮可将2'-脱氧鸟苷（dGuo）转化为2'-脱氧氧杂鸟苷（dOxo）[铃木，T.，山冈，R.，西，M.，井手，H.，&牧野，K.（1996年）《美国化学会志》118，2515 - 2516]。在本研究中，我们测定了dOxo的N - 糖苷键对自发水解的敏感性及其碱基配对稳定性，以评估dOxo作为DNA中新损伤的生物学意义。当从经亚硝酸处理的d（T5GT6）中分离出的寡脱氧核苷酸d（T5OT6）（O = dOxo）在pH 4.0和70℃下孵育时，dOxo的N - 糖苷键以一级速率常数发生水解。将该速率常数与dGuo和dXao的速率常数进行比较表明，dOxo在d（T5GT6）中的N - 糖苷键与dGuo的一样稳定，并且比同时由亚硝酸和一氧化氮产生的损伤2'-脱氧黄苷（dXao）的N - 糖苷键水解慢44倍。为了估计碱基配对稳定性，在中性pH下测量了d（T5OT6）.d（A6NA5）（N = A、G、C和T）双链体的紫外熔解曲线。对于N = A、G、C和T，得到的熔解温度（Tm）值分别为15.3、14.1、19.3和16.3℃，这些值远低于在相同位置含有G.C对的完整双链体[d（T5GT6）.d（A6CA5），Tm = 32.8℃]，但与d（T5XT6）.d（A6NA5）（X = dXao，Tm = 14.8 - 22.3℃）的相当。含有dOxo的四种双链体的圆二色光谱表明在低温下完整双链体的结构得以保留。紫外和核磁共振pH滴定研究表明开环和闭环平衡的pKa为9.4，这意味着dOxo在生理pH下处于闭环形式。这种结构在几何形状上似乎不适合与特定互补碱基形成氢键，因此导致所有互补碱基的Tm值同样较低。因此，这些结果表明dOxo作为一种新的DNA损伤，可能在细胞的诱变事件中具有重要且独特的作用。
