Povirk L F, Goldberg I H
Proc Natl Acad Sci U S A. 1982 Jan;79(2):369-73. doi: 10.1073/pnas.79.2.369.
When the nonprotein chromophore of neocarzinostatin was allowed to react with either calf thymus DNA or poly(dA-dT) . poly(dA-dT) in the presence of 2-mercaptoethanol and the DNA was precipitated with ethanol, 5% of the fluorescence attributable to the naphthalene rings of the chromophore coprecipitated with the DNA. Most of this fluorescence remained attached to DNA through successive reprecipitations, suggesting formation of covalent adducts between chromophore and DNA. Enzymatically digested poly(dA-dT) . poly(dA-dT)-chromophore adduct contained, in addition to deoxyadenosine and thymidine, several highly fluorescent hydrophobic products, separable by reverse-phase chromatography, all of which contained both adenine and thymine radiolabel, as well as chromophore radiolabel. One such product consistently had twice as much thymine as adenine, suggesting a structure chromophore-d(TpApT), in which the attached chromophore rendered both phosphodiester bonds refractory to endonuclease S1. This adduct fragment was completely hydrolyzed at pH 12, releasing adenine, 3'-dTMP, and 5'-dTMP. At pH 7, the adduct fragment slowly released chromophore and 3'-dTMP with parallel kinetics, leaving a modified d(ApT), which was cleaved by snake venom phosphodiesterase to yield 5'-dTMP and a modified deoxyadenosine. These hydrolysis patterns are unlike those of any previously characterized base or phosphotriester DNA adduct but rather indicate an altered deoxyadenosine sugar. The formation of adducts containing a modified deoxyribose suggests that deoxyribose may be the site of covalent chromophore attachment. Alteration of this same site, possibly the 5'-carbon of the sugar moiety, may account for the extreme lability of the phosphodiester bond.
当新制癌菌素的非蛋白质发色团在2-巯基乙醇存在的情况下与小牛胸腺DNA或聚（dA-dT）·聚（dA-dT）反应，并且DNA用乙醇沉淀时，发色团萘环产生的荧光有5%与DNA共沉淀。通过连续再沉淀，大部分这种荧光仍与DNA相连，这表明发色团与DNA之间形成了共价加合物。经酶消化的聚（dA-dT）·聚（dA-dT）-发色团加合物，除了脱氧腺苷和胸苷外，还含有几种高度荧光的疏水产物，可通过反相色谱法分离，所有这些产物都含有腺嘌呤和胸腺嘧啶放射性标记以及发色团放射性标记。其中一种这样的产物胸腺嘧啶含量始终是腺嘌呤的两倍，这表明其结构为发色团-d（TpApT），其中连接的发色团使两个磷酸二酯键对内切核酸酶S1具有抗性。该加合物片段在pH 12时完全水解，释放出腺嘌呤、3'-dTMP和5'-dTMP。在pH 7时，加合物片段以平行动力学缓慢释放发色团和3'-dTMP，留下一种修饰的d（ApT），它被蛇毒磷酸二酯酶切割产生5'-dTMP和一种修饰的脱氧腺苷。这些水解模式与任何先前表征的碱基或磷酸三酯DNA加合物的水解模式不同，而是表明脱氧腺苷糖发生了改变。含有修饰脱氧核糖的加合物的形成表明脱氧核糖可能是共价发色团附着的位点。同一位点（可能是糖部分的5'-碳）的改变可能解释了磷酸二酯键的极度不稳定性。
