Effective Utilization of N²-Ethyl-2‘-deoxyguanosine Triphosphate during DNA Synthesis Catalyzed by Mammalian Replicative DNA Polymerases

Abstract

Acetaldehyde is produced by metabolic oxidation of ethanol after drinking alcoholic beverages. This agent reacts with nucleosides and nucleotides, resulting in the formation of N2-ethyl-guanine residues. N2-ethyl-2'-deoxyguanosine (N2-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. N2-Ethyl-2'-deoxyguanosine triphosphate (N2-ethyl-dGTP) was chemically synthesized. The utilization of N2-ethyl-dGTP during DNA synthesis was determined by steady-state kinetic studies. N2-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 N2-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 N2-ethyl-dGTP was only 37 times less than that of dGTP. The chain extension from dC:N2-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 N2-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 N2-ethyl-dG adducts into DNA may cause mutations and may be related to the development of alcohol- and acetaldehyde-induced human cancers.

Risk factors and mechanisms of hepatocarcinogenesis with special emphasis on alcohol and oxidative stress

“…The occurrence of stable DNA adducts has been shown in different organs of alcohol-fed rodents and in leukocytes of alcoholics (Fang and Vaca, 1997). It has been shown that the major stable DNA adduct N 2 -ethyl desoxyguanosine (N2-Et-dG) indeed serves as a substrate of eukaryotic DNA polymerase (Matsuda et al, 1999). More recently, another DNA adduct of acetaldehyde namely 1,N 2 -propano-desoxyguanosine (PdG) has been identified (Brooks and Theruvathu, 2005).…”

Structure-Function Relationships in Miscoding by Sulfolobus solfataricus DNA Polymerase Dpo4: GUANINE N2,N2-DIMETHYL SUBSTITUTION PRODUCES INACTIVE AND MISCODING POLYMERASE COMPLEXES

“…Pyridyloxobutyl-derived N 2 -alkyl G adducts have been detected in rats after chronic treatment with NЈ-nitrosonornicotine (31). N 2 -EtG was detected in granulocyte and lymphocyte DNA and urine of alcoholic patients (32,33), and misincorporation opposite N 2 -EtG by Escherichia coli DNA polymerase I/Klenow fragment (exonuclease Ϫ ) has been reported (22). Crotonaldehyde-and acetaldehyde-derived 1,N 2 -propanodeoxyguanosine adducts have also been characterized in human tissue DNA (25).…”

Technology and methodology of DNA adducts analysis

“…同样在体内一些还原性小分子或酶, 如维生 素C和谷胱苷肽(GSH)催化作用下能够将EtidG部分转 化为EthdG [98,99] ; 然而, 对于DNA中EtidG加合物的检测 需要通过化学还原的方法在体外将EtidG定量转化为 EthdG, 该过程通过加入化学还原剂NaBH4或NaBH3CN 来实现 [97,100] . 基于以上方法, Wang等 [97] 在没有经过外 源乙醛暴露的人类肝脏基因组DNA中检测到相对丰 度较高的EthdG加合物, 约为0.1个/10 6 个核苷酸, 表明 了肝脏细胞正常代谢过程中乙醛的产生及内源性乙 醛-DNA加合物的生成.…”