Effective utilization of N2-ethyl-2′-deoxyguanosine triphosphate during DNA synthesis catalyzed by mammalian replicative DNA polymerases
Effective utilization of N2-ethyl-2′-deoxyguanosine triphosphate during DNA synthesis catalyzed by mammalian replicative DNA polymerases
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DOI：
10.1021/bi982134j
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发表时间：
1999-01-19
期刊：
影响因子：
2.9
通讯作者：
Shibutani, S
中科院分区：
文献类型：
--
作者：
Matsuda, T;Terashima, I;Shibutani, S
乙醛是饮用酒精饮料后乙醇代谢氧化产生的。该试剂与核苷和核苷酸反应，形成n -2-乙基鸟嘌呤残基。n -2-乙基-2'-脱氧鸟苷（n -2-乙基- dg）加合物在酒精患者淋巴细胞DNA中被检测[Fang， J. L., Vaca， C. E. (1997) Carcinogenesis 18, 627-632]。因此，预计核苷酸库也会被乙醛修饰。化学合成了n -2-乙基-2'-脱氧鸟苷三磷酸（n -2-乙基- dgtp）。通过稳态动力学研究确定了n -2-乙基- dctp在DNA合成过程中的利用。在哺乳动物DNA聚合酶α和δ催化的反应中，n -2-乙基- dgtp有效地与模板dC相结合。当使用pol α时，n -2-乙基-dGTP的插入频率比dGTP低400倍，但比氧化损伤核苷酸7,8-二氢-8-氧-2'-脱氧鸟苷三磷酸（8-氧-dGTP）的插入频率高320倍。采用pol δ法，n -2-乙基-dGTP的插入频率仅为dGTP的37倍。dC: n -2-乙基-dG对的链延伸速度更快，pol α和pol δ的链延伸频率分别仅为dC:dG对的3.8倍和6.3倍。我们还发现，在收集尿液前一周不饮酒的健康志愿者的尿液样本中可以检测到n -2-乙基- dg。这表明即使不喝酒精饮料，人类也会持续暴露在乙醛中。将n -2-乙基- dg加合物掺入DNA可能导致突变，并可能与酒精和乙醛诱导的人类癌症的发生有关。
Acetaldehyde is produced by metabolic oxidation of ethanol after drinking alcoholic beverages. This agent reacts with nucleosides and nucleotides, resulting in the formation of N-2-ethyl-guanine residues. N-2-ethyl-2'-deoxyguanosine (N-2-ethyl-dG) adduct has been detected in the lymphocyte DNA of alcoholic patients [Fang, J. L., and Vaca, C. E. (1997) Carcinogenesis 18, 627-632]. Thus, the nucleotide pool is also expected to be modified by acetaldehyde. N-2-Ethyl-2'-deoxyguanosine triphosphate (N-2-ethyl-dGTP) was chemically synthesized. The utilization of N-2-ethyl-dCTP during DNA synthesis was determined by steady-state kinetic studies. N-2-Ethyl-dGTP was efficiently incorporated opposite template dC in reactions catalyzed by mammalian DNA polymerase alpha and delta. When pol alpha was used, the insertion frequency of N-2-ethyl-dGTP was 400 times less than that of dGTP, but 320 times higher than that of 7,8-dihydro-8-oxo-2'-deoxyguanosine triphosphate (8-oxo-dGTP), an oxidative damaged nucleotide. Using pol delta, the insertion frequency of N-2-ethyl-dGTP was only 37 times less than that of dGTP. The chain extension from dC:N-2-ethyl-dG pair occurred much more rapidly: the extension frequencies for pol alpha and pol delta were only 3.8 times and 6.3 times, respectively, lower than that of dC:dG pair. We also found that N-2-ethyl-dG can be detected in urine samples obtained from healthy volunteers who had abstained from drinking alcohol for 1 week before urine collection. This indicates that humans are exposed constantly to acetaldehyde even without drinking alcoholic beverages. Incorporation of N-2-ethyl-dG adducts into DNA may cause mutations and may be related to the development of alcohol- and acetaldehyde-induced human cancers.