Sites of termination of in vitro DNA synthesis on ultraviolet- and N-acetylaminofluorene-treated phi X174 templates by prokaryotic and eukaryotic DNA polymerases

Abstract

In vitro DNA synthesis on a phi X174 template primed with a restriction fragment and catalyzed by the Escherichia coli DNA polymerase I large (Klenow) fragment (pol I) terminates at the nucleotide preceding a site that has been altered by ultraviolet irradiation or treatment with N-acetylaminofluorene. Termination on ultraviolet-irradiated templates is similar when synthesis is catalyzed by E. coli DNA polymerase III holoenzyme (pol III), phage T4 DNA polymerase, a polymerase alpha from human lymphoma cells, or avian myeloblastosis virus reverse transcriptase. 3' leads to 5' exonuclease activity cannot be detected in the reverse transcriptase and DNA polymerase alpha preparations. On N-acetylaminofluorene templates, pol I, pol III, and T4 polymerase reactions terminate immediately preceding the lesion, whereas reverse transcriptase-catalyzed reactions and, at some positions in the sequence, polymerase alpha-catalyzed reactions terminate at the site of the lesion. Substitution of Mn2+ for Mg2+ changes the pattern of pol I-catalyzed termination sites. The data suggest that termination is a complicated process that does not depend exclusively on the 3' leads to 5' exonuclease activity associated with many polymerases.