DNA-POLYMERASE ACTION ON AN OLIGONUCLEOTIDE CONTAINING A SITE-SPECIFICALLY LOCATED N-(DEOXYGUANOSIN-8-YL)-1-AMINOPYRENE

A 25mer oligonucleotide containing a single N-(deoxyguanosin-8-yl)-1-a minopyrene (dG(AP)), the major DNA adduct formed by reductively activa ted l-nitropyrene, was synthesized, The adduct was located at nucleoti de 21 from the 3' end, DNA synthesis on this template by human DNA pol ymerases alpha and beta, HIV reverse transcriptase, Sequenase (version 2.0) and Klenow fragment of DNA polymerase I was strongly blocked at the nucleotide 3' to the adduct site, Only when a 3'-->5' exonuclease- deficient Klenow fragment was used was incorporation of a nucleotide o pposite the adduct observed. Nevertheless, extension beyond the adduct site did not occur to a significant extent, Only a relatively small p roportion of full-length product (<5%) was detected, In the presence o f Mn2+, the efficiency of bypass with this polymerase increased, When a 20mer primer was elongated in the presence of only one nucleotide tr iphosphate, deoxycytidylic acid was preferentially incorporated opposi te the adduct. Deoxycytidine opposite the adduct was also preferred wh en a set of 2lmer primers (containing each of the four nucleotides opp osite dG(AP)) were elongated to a full-length product in the presence of all four deoxynucleotide triphosphates. In order to confirm these r esults, extension of a 15mer primer was carried out with all four deox ynucleotide triphosphates and the products were isolated. Maxam-Gilber t sequencing of each elongation product showed that primer extension o ccurred in an error-free manner. We conclude that dG(AP) is a strong b lock of DNA replication, However, when translesion synthesis occurs, i t is largely accurate.