Escherichia coli polymerase I can use O2-methyldeoxythymidine or O4-methyldeoxythymidine in place of deoxythymidine in primed poly(dA-dT).poly(dA-dT) synthesis.
Escherichia coli polymerase I can use O2-methyldeoxythymidine or O4-methyldeoxythymidine in place of deoxythymidine in primed poly(dA-dT).poly(dA-dT) synthesis.
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大肠杆菌聚合酶 I 可以在引发的聚（dA-dT）.聚（dA-dT）合成中使用 O2-甲基脱氧胸苷或 O4-甲基脱氧胸苷代替脱氧胸苷。
DOI：
10.1073/pnas.80.16.4884
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发表时间：
1983
影响因子：
11.1
通讯作者：
Kuśmierek,JT
中科院分区：
文献类型：
--
作者：
Singer,B;Sági,J;Kuśmierek,JT
O2-和O4-烷基脱氧胸腺嘧啶核苷是N-亚硝基烷基化试剂与核酸在体内外反应生成的四种O-烷基修饰的衍生物之一。我们发现O2-和O4-甲基-dTTP在交替多聚(da-dT)引发的DNA合成中都可以替代dTTP。经高效液相色谱分析，新合成的聚合物中有高达22%的嘧啶是O-甲基脱氧胸苷。在没有dTTP的情况下，几乎没有观察到聚合物的合成。然而，O-甲基-dTTP并不抑制dATP和dTTP的聚合。以较高的产率合成了含O2-或O4-甲基脱氧胸苷的聚合物，保留了交替聚(da-dt)的二级结构。不同条件下的热转变数据表明了这一点。相反，由烷基亚硝脲甲基化或乙基化到总改性量小于4%的聚(da-dt).聚(da-dt)的结构明显不稳定。O2-和O4-甲基脱氧胸苷都不能与腺苷形成一个以上的氢键。含有这些修饰胸苷的聚合物的二级结构不变，这表明堆积相互作用在螺旋稳定中肯定发挥了主要作用。O-烷基脱氧胸腺嘧啶核苷可能是由N-亚硝基致癌物在细胞内反应形成的。我们已经证明，三磷酸盐可以被大肠杆菌DNA聚合酶I用作dTTP。被掺入的O4-甲基-DT在转录和合成中都会导致G的错配。当O2-甲基-DT存在时，错误结合的结果较少，但肯定的。
O2-and O4-alkyldeoxythymidine are among the four O-alkyl base-modified derivatives produced by the reaction of N-nitroso alkylating agents with nucleic acids in vitro and in vivo. We find that both O2- and O4-methyl-dTTP can substitute for dTTP in alternating poly(dA-dT)-primed DNA synthesis. Up to 22% of the pyrimidines in the newly synthesized polymer were found by HPLC analysis to be O-methyldeoxythymidine. Little polymer synthesis was observed in the absence of dTTP. However, the O-methyl-dTTPs did not inhibit polymerization of dATP and dTTP. Polymers containing O2- or O4-methyldeoxythymidine were obtained in good yield, retaining the secondary structure of alternating poly(dA-dT). This was shown by the data for thermal transition under different conditions. In contrast, poly(dA-dT).poly(dA-dT) methylated or ethylated to less than 4% total modification by alkylnitrosoureas had a distinctly less stable structure. Neither O2- nor O4-methyldeoxythymidine can form more than one hydrogen bond with adenosine. The unchanged secondary structure of polymers containing these modified thymidines indicates that stacking interactions must play a major role in helix stabilization. O-Alkyldeoxythymidine may be formed by N-nitroso carcinogens that react intracellularly. We have shown that the triphosphates can be utilized by Escherichia coli DNA polymerase I as dTTP. The incorporated O4-methyl-dT causes misincorporation of G, both in transcription and synthesis. When O2-methyl-dT is present, less, but definite, misincorporation results.