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首页> 外文期刊>Biochemistry >Miscoding potential of the N~2-ethyl-2'-deoxyguanosine DNA adduct by the exonuclease-free klenow fragment of escherichia coli DNA polymerase I
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Miscoding potential of the N~2-ethyl-2'-deoxyguanosine DNA adduct by the exonuclease-free klenow fragment of escherichia coli DNA polymerase I

机译：大肠杆菌DNA聚合酶I的无核酸外切酶klenow片段导致N〜2-乙基-2'-脱氧鸟苷DNA加合物的错误编码潜力

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摘要

Acetaldehyde, a major metabolite of ethanol, reacts with dG residues in DNA, resulting in the formation of the N2-ethyl-2’-deoxyguanosine (N2-Et-dG) adduct. This adduct has been detected in lymphocyte DNA of alcohol abusers. To explore the miscoding property of the N2-Et-dG DNA adduct, phosphoramidite chemical synthesis was used to prepare site-specifically modified oligodeoxynucleotides containing a single N2-Et-dG. These N2-Et-dG-modified oligodeoxynucleotides were used as templates for primer extension reactions catalyzed by the 3’ 5’ exonuclease-free (exo) Kienow fragment of Escherichia coli DNA polymerase I. The primer extension was retarded one base prior to the N2-Et-dG lesion and opposite the lesion; however, when the enzyme was incubated for a longer time or with increased amounts of this enzyme, full extension occurred. Quantitative analysis of the fully extended products showed the preferential incorporation of dGMP and dCMP opposite the N2-Et-dG lesion, accompanied by a small amounts of dAMP and dTMP incorporation and one- and two-base deletions. Steady-state kinetic studies were also performed to determine the frequency of nucleotide insertion opposite the N2-Et-dG lesion and chain extension from the 3’ terminus from the dNN2-Et-dG (N is C, A, G, or T) pairs. These results indicate that the N2-Et-dG DNA adduct may generate G —~ C transversions in living cells. Such a mutational spectrum has not been detected with other methylated dG adducts, including 8-methyl-2’-deoxyguanosine, O6-methyl-2’-deoxyguanosine, and N2-methyl-2’-deoxyguanosine. In addition, N2-ethyl-2’-deoxyguanosine triphosphate (N2-Et-dGTP) was efficiently incorporated opposite a template dC during DNA synthesis catalyzed by the exo Klenow fragment. The utilization of N2-Et-dGTP was also determined by steady-state kinetic studies. N2-Et-dG DNA adducts are also formed by the incorporation of N2-Et-dGTP into DNA and may cause mutations, leading to the development of alcohol- and acetaldehyde-induced human cancers.
机译：乙醛是乙醇的主要代谢产物，它与DNA中的dG残基反应，导致形成N2-乙基-2'-脱氧鸟苷（N2-Et-dG）加合物。在酒精滥用者的淋巴细胞DNA中已检测到这种加合物。为了探索N2-Et-dG DNA加合物的误编码特性，使用亚磷酰胺化学合成制备了含有单个N2-Et-dG的位点特异性修饰的寡脱氧核苷酸。这些N2-Et-dG修饰的寡脱氧核苷酸被用作模板，用于由大肠杆菌DNA聚合酶I的无3'5'核酸外切酶（exo）Kienow片段催化的引物延伸反应。在N2之前，引物延伸被延迟了一个碱基。 -Et-dG病变和相对病变;然而，当酶孵育更长的时间或与增加量的酶一起孵育时，会发生完全延伸。完全扩展的产物的定量分析显示，与N2-Et-dG病灶相对的是dGMP和dCMP的优先掺入，伴随有少量dAMP和dTMP掺入以及一碱基和两碱基缺失。还进行了稳态动力学研究，以确定与N2-Et-dG损伤相对的核苷酸插入频率和dNN2-Et-dG的3'末端的链延伸（N为C，A，G或T）对。这些结果表明，N2-Et-dG DNA加合物可能在活细胞中产生G C转位。其他甲基化的dG加合物（包括8-甲基-2'-脱氧鸟苷，O6-甲基-2'-脱氧鸟苷和N2-甲基-2'-脱氧鸟苷）没有检测到这种突变谱。另外，在由外Klenow片段催化的DNA合成过程中，N2-乙基-2′-脱氧鸟苷三磷酸酯（N2-Et-dGTP）被有效地掺入模板dC对面。 N2-Et-dGTP的利用还通过稳态动力学研究确定。 N2-Et-dG DNA加合物也是通过将N2-Et-dGTP掺入DNA形成的，并可能引起突变，从而导致酒精和乙醛诱导的人类癌症的发展。

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