6533b824fe1ef96bd128139c

RESEARCH PRODUCT

DNA Polymerase Action on Oligonucleotide Templates from Human Ha-rasProtooncogene Containing N6-Deoxyadenosine Adducts Derived from Trans Addition of (+)- and (-)-anti-Benzo[c]phenanthrene-3,4-dihydrodiol 1,2-epoxides at Codon 61

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Abstract In the present work we have used a DNA polymerase assay to investigate the primer extension with T7 DNA polymerase (Sequenase 2.0) and the Klenow fragment of Escherichia coli DNA polymerase I (exo − KF) on chemically synthesized 21mer templates representing partial sequences of the human Ha-ras protooncogene with site-specifically positioned trans-N 6-dA adducts of (-)- (adduct 1) and (+)-anti-benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxides (adduct 2) at codon 61 (CA∗G; A∗ indicates the adducted position). With Sequenase 2.0 a complete block of primer extension opposite both adduct 1 and 2 was noted using a 10mer primer reaching the (n-1)-position of the adduct. A detailed analysis revealed that Sequenase 2.0 exclusively allowed the misincorporation of dAMP opposite both adducts. In contrast, exo − KF allowed a significant full-length extension of the primer beyond adduct 2, whereas this process occured only to a very minor extent for adduct 1. Correspondingly, a more intensive block of the pol...