Richardson F C, Richardson K K
Toxicology Division, Eli Lilly and Co., Greenfield, Indiana 46140.
Mol Carcinog. 1991;4(2):162-8. doi: 10.1002/mc.2940040212.
O4-Alkyldeoxythymidines have been extensively studied for their ability to cause mutations and to induce cancer. Since these adducts can change DNA conformation, they may also have a more immediate effect of altering DNA-protein interactions. To address this issue, the effects of these adducts on restriction enzyme activity were examined. Oligodeoxyribonucleosides containing O4-ethyldeoxythymidine (O4-EtdT) or O4-methyldeoxythymidine (O4-MedT) at a unique site within the sequence 5'-GAATGGATCCTAATGAGATC-3' were constructed by automated DNA synthesis. This sequence contains the recognition site for various restriction enzymes. These oligomers were annealed to various complementary strands and digested with restriction enzymes: BamHI or BstI (GGATCC); Sau3A, NdeII, or MboI (GATC); DpnI (GmATC); and BstYI, MflI, or XhoII (PuGATCPy). Analysis of the digests demonstrated that the presence of either O4-EtdT or O4-MedT abolished the ability of XhoII, MboI, MflI, or NdeII to cut at the restriction site. DpnI failed to cut any of the oligomers. BamHI, Sau3A, BstI, and BstYI exhibited alterations in cutting specificity depending upon the oligomers used. These results demonstrated that O4-alkyldeoxythymidine adducts alter DNA-restriction enzyme interactions in a protein- and sequence-dependent manner. Because of the importance of natural methylation in genetic regulation, it is possible that aberrant methylation in the form of DNA adducts could also alter protein-DNA interactions in cells exposed to DNA-modifying agents.
O4-烷基脱氧胸苷因其导致突变和诱发癌症的能力而受到广泛研究。由于这些加合物会改变DNA构象，它们可能还会对改变DNA-蛋白质相互作用产生更直接的影响。为了解决这个问题，研究了这些加合物对限制酶活性的影响。通过自动DNA合成构建了在序列5'-GAATGGATCCTAATGAGATC-3'内的一个独特位点含有O4-乙基脱氧胸苷（O4-EtdT）或O4-甲基脱氧胸苷（O4-MedT）的寡脱氧核糖核苷。该序列包含多种限制酶的识别位点。将这些寡聚物与各种互补链退火，并用限制酶进行消化：BamHI或BstI（GGATCC）；Sau3A、NdeII或MboI（GATC）；DpnI（GmATC）；以及BstYI、MflI或XhoII（PuGATCPy）。对消化产物的分析表明，O4-EtdT或O4-MedT的存在消除了XhoII、MboI、MflI或NdeII在限制位点切割的能力。DpnI无法切割任何一种寡聚物。BamHI、Sau3A、BstI和BstYI根据所使用的寡聚物表现出切割特异性的改变。这些结果表明，O4-烷基脱氧胸苷加合物以蛋白质和序列依赖性方式改变DNA-限制酶相互作用。由于天然甲基化在基因调控中的重要性，以DNA加合物形式存在的异常甲基化也有可能改变暴露于DNA修饰剂的细胞中的蛋白质-DNA相互作用。
