THE ROLE OF 3-HYDROXYETHYLDEOXYURIDINE IN MUTAGENESIS BY ETHYLENE-OXIDE

Ethylene oxide, a direct-acting mutagen and carcinogen, produces 3-hyd roxyethyldeoxyuridine (3-HE-dU) after initial alkylation at N3 of de, followed by rapid hydrolytic deamination. The significance of formatio n 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 si te, was constructed. DNA products, synthesized on the site-modified te mplate, were analyzed and mutagenic bypass at 3-HE-dU estimated. The 3 -HE-dU lesion blocked DNA replication by the Klenow fragment of Escher ichia coil polymerase I (Rf 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' e xonuclease proofreading activity, for Hf Pol I and T7 Pol, respectivel y, facilitated DNA synthesis past 3-HE-dU. The bypass synthesis by Kf Poll (exo(-)) was 60% and 90% by T7 Pol (exo(-)). These results sugges t 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 nucleot ide incorporated opposite 3-HE-dU is not excised and remains in positi on long enough for extension to occur. During post-lesion synthesis, b oth dA and dT were incorporated opposite 3-HE-dU. Since 3-HE-dU is der ived 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.