Miscoding potential of the N2-ethyl-2'-deoxyguanosine DNA adduct by the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I.
Acetaldehyde, a major metabolite of ethanol, reacts with dG residues in DNA, resulting in the formation of the N(2)-ethyl-2'-deoxyguanosine (N(2)-Et-dG) adduct. This adduct has been detected in lymphocyte DNA of alcohol abusers. To explore the miscoding property of the N(2)-Et-dG DNA adduct, phosphoramidite chemical synthesis was used to prepare site-specifically modified oligodeoxynucleotides containing a single N(2)-Et-dG. These N(2)-Et-dG-modified oligodeoxynucleotides were used as templates for primer extension reactions catalyzed by the 3' --> 5' exonuclease-free (exo(-)) Klenow fragment of Escherichia coli DNA polymerase I. The primer extension was retarded one base prior to the N(2)-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 N(2)-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 N(2)-Et-dG lesion and chain extension from the 3' terminus from the dN.N(2)-Et-dG (N is C, A, G, or T) pairs. These results indicate that the N(2)-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, O(6)-methyl-2'-deoxyguanosine, and N(2)-methyl-2'-deoxyguanosine. In addition, N(2)-ethyl-2'-deoxyguanosine triphosphate (N(2)-Et-dGTP) was efficiently incorporated opposite a template dC during DNA synthesis catalyzed by the exo(-) Klenow fragment. The utilization of N(2)-Et-dGTP was also determined by steady-state kinetic studies. N(2)-Et-dG DNA adducts are also formed by the incorporation of N(2)-Et-dGTP into DNA and may cause mutations, leading to the development of alcohol- and acetaldehyde-induced human cancers.
Terashima
I
I
Laboratory of Chemical Biology, Department of Pharmacological Sciences, State University of New York at Stony Brook, 11794-8651, USA.
Matsuda
T
T
Fang
T W
TW
Suzuki
N
N
Kobayashi
J
J
Kohda
K
K
Shibutani
S
S
D018736:DNA Adducts / Q000235:genetics* / Q000378:metabolism; D004256:DNA Polymerase I / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005090:Exodeoxyribonucleases / Q000378:metabolism; D005815:Genetic Code; D007364:Intercalating Agents / Q000378:metabolism; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D013698:Templates, Genetic
D018736:DNA Adducts; D017931:DNA Primers; D004269:DNA, Bacterial; D003848:Deoxyguanine Nucleotides; D007364:Intercalating Agents; C117229:N(2)-ethyl-2'-deoxyguanosine triphosphate; D009838:Oligodeoxyribonucleotides; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases