Miscoding potential of the N-2-ethyl-2 '-deoxyguanosine DNA adduct by the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I

Citation
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
Citations number
29
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMISTRY
ISSN journal
0006-2960 → ACNP
Volume
40
Issue
13
Year of publication
2001
Pages
4106 - 4114
Database
ISI
SICI code
0006-2960(20010403)40:13<4106:MPOTN'>2.0.ZU;2-A
Abstract
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.