Abstract
Ethylene oxide, a direct-acting mutagen and carcinogen, produces 3-hydroxyethyldeoxyuridine (3-HE-dU) after initial alkylation at N3 of dC, followed by rapid hydrolytic deamination. The significance of formation of 3-HE-dU in DNA was investigated by in vitro DNA replication of 3-HE-dU. A 55-nucleotide DNA template, containing 3-HE-dU at a single site, was constructed. DNA products, synthesized on the site-modified template, were analyzed and mutagenic bypass at 3-HE-dU estimated. The 3-HE-dU lesion blocked DNA replication by the Klenow fragment of Escherichia coli polymerase I (Kf Pol I) and bacteriophage T7 polymerase (T7 Pol) 3' to 3-HE-dU and after incorporating a nucleotide opposite 3-HE-dU. DNA synthesis past 3-HE-dU was negligible (< 3%). Substitution of Kf Pol I (exo-) and T7 Pol (exo-), polymerases lacking 3'-->5' exonuclease proofreading activity, for Kf Pol I and T7 Pol, respectively, facilitated DNA synthesis past 3-HE-dU. The bypass synthesis by Kf Pol I (exo-) was 60% and 90% by T7 Pol (exo-). These results suggest that the 3-HE-dU lesion could be bypassed, but that the extension at 3-HE-dU is rate-limiting. In the absence of proofreading, the nucleotide incorporated opposite 3-HE-dU is not excised and remains in position long enough for extension to occur. During post-lesion synthesis, both dA and dT were incorporated opposite 3-HE-dU. Since 3-HE-dU is derived from dC alkylation by ethylene oxide, incorporation of dA and dT opposite 3-HE-dU implicates this lesion in G.C-->A.T and G.C-->T.A mutagenesis.
Highlights
Ethylene oxide,a direct-acting mutagen and carcino- Ethylene oxide is genotoxic [2,8,9] and causes mutationisn gen,produces 3-hydroxyethyldeoxyuridine (3-HE-dU) a number of biological systems including Salmonella typhiafter initial alkylation at N3ofdC, followed by rapid murium
Since 3-HE-dU is derived from ethylene oxide-induced 3-HE
DC, incorporation of dA and dT opposite 3-HE-dU implicates this lesion in G-C + A-T and G C + T.A mutagenesis
Summary
3-~2-Acetoxyethyli-5'-O-f4,4'-dimethoxytrityl)-2'-deoxyuridine~3'-(c2e-ntration of 15 m ~T.he reaction volume was reduced to about 30 p1 cyanoethyl-N,N-diisopropylphosphoramiditej (DMTr-(3-AEjdU-phos- using a SpeedVac concentrator (Savant), mixed with 50 pI of formamide phorumidite; Fig. I)-The method described fonrormal nucleosides[46] dye mix [49], and the mixture heated at 100 "C for 3 min before sepaand adapted for modified nucleosides 3-Et-dT [36] and O'-ethyldeoxy- ration on a 16%polyacrylamide, M urea gel (1mm thick). Diluted with 15 mlof anhydrous ethyl acetate and transferred to a mmol) 17-nucleotide primer was annealed t o the site-modified 55-nuseparatory funnel containing 20 ml of ethyl acetate and 50 mlof satu- cleotidetemplate as described previously (36T).he yield of the resulting rated brine. The resulting gum was DNA PolymeraseReaction-The reaction mixture (7 111) contained 10 purified by silica gelcolumn (4 cm x 23cm) chromatography using mM HEPES buffer, pH 7.5,5 mM MgCl,, 2 mM DTT, 0.1 pmol of primed solvent system I11 as aneluent.
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