I. Terashima et al., Miscoding potential of the N-2-ethyl-2 '-deoxyguanosine DNA adduct by the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I, BIOCHEM, 40(13), 2001, pp. 4106-4114
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-de DNA adduct, phosphoram
idite chemical synthesis was used to prepare site-specifically modified oli
godeoxynucleotides 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 Es
cherichia 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 e
nzyme was incubated for a longer time or with increased amounts of this enz
yme, full extension occurred. Quantitative analysis of the fully extended p
roducts showed the preferential incorporation of dGMP and dCMP opposite the
N-2-Et-dG lesion, accompanied by a small amounts of dAMP and dTMP incorpor
ation and one- and two-base deletions. Steady-state kinetic studies were al
so performed to determine the frequency of nucleotide insertion opposite th
e 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-d
G DNA adduct may generate G --> C transversions in living cells. Such a mut
ational spectrum has not been detected with other methylated dG adducts, in
cluding 8-methyl-2'-deoxyguanosine, O-6-methyl-2'-deaxyguanosine, and N-2-m
ethyl-2'-deoxyguanosine. In addition, N-2-ethyl-2'-deoxyguanosine triphosph
ate (N-2-Et-dGTP) was efficiently incorporated opposite a template dC durin
g 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 D
NA 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 acetaldehy
de-induced human cancers.