The role of 3-hydroxyethyldeoxyuridine in mutagenesis by ethylene oxide.
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.
Bhanot
O S
OS
Department of Environmental Medicine, New York University Medical Center, New York 10016.
Singh
U S
US
Solomon
J J
JJ
D001483:Base Sequence; D004247:DNA / Q000187:drug effects; D004249:DNA Damage; D004261:DNA Replication / Q000187:drug effects; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000378:metabolism; D005027:Ethylene Oxide / Q000633:toxicity*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009153:Mutagens / Q000633:toxicity*
D009153:Mutagens; C090624:3-hydroxyethyldeoxyuridine; D004247:DNA; D005027:Ethylene Oxide; D003857:Deoxyuridine