Base incorporation and extension at a site-specific ethenocytosine by Escherichia coli DNA polymerase I Klenow fragment

Ethenocytosine (_ε{lunate}C) is a highly mutagenic exocyclic DNA lesion induced by carcinogens vinyl chloride and urethane. We have examined base incorporation and extension at a site-specific _ε{lunate}C residue by a quantitative gel electrophoretic assay using an exonuclease-deficient version of Escherichia coli DNA polymerase I (Klenow fragment) as the model enzyme. The data show that the K_m for incorporation of adenine or thymine opposite _ε{lunate}C by is about 5 orders of magnitude higher than that for the incorporation of guanine opposite normal cytosine. The K_M for base extension past _ε{lunate}C:A and _ε{lunate}C:T pairs is 1-2 orders of magnitude higher than that observed for a C:G pair. Although adenine misinsertion is favored over that of thymine, base extension occurs more rapidly when the base incorporated opposite _ε{lunate}C is thymine.