Evidence for reduced copying fidelity of DNA polymerases α, δ, and ε from Novikoff hepatoma cells

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Abstract
To investigate whether or not DNA polymerases α, δ, and ε from tumor cells have acquired properties that might be responsible for mutations found in tumor development, we investigated copying fidelities of DNA polymerases α, δ, and ε from the highly malignant Novikoff hepatoma cells and compared them to the corresponding enzymes from normal rat liver. DNA polymerases were purified more than 300-fold by three chromatographic steps. Copying fidelity was studied using steady-state kinetics and an 18-mer oligonucleotide primed with a 12-mer (13-mer for extension experiments) as DNA primer-template. Three experimental approaches were chosen: i) extension of DNA primers with mismatched 3’-OH ends opposite dGMP, ii) DNA insertion of nucleotides opposite m6G in the template and iii) extension of DNA primers with mismatched 3’-OH ends opposite m6G. i) Extension of DNA primers with mismatched 3’-OH ends opposite dGMP. DNA primer templates containing G:T and G:A mispairs at the 3’-OH position of the primer were easily extended by DNA polymerases α, δ and ε from both normal rat liver and Novikoff hepatoma cells. The G:G mismatch was elongated with low efficiency. Notably, DNA polymerase α from Novikoff hepatoma cells extended G:A and G:G mismatches significantly faster than the enzyme from normal cells. ii) Insertion of nucleotides opposite m 6 G. DNA polymerases α, δ, and ε from normal rat liver preferably catalyzed incorporation of dAMP opposite m6G at dNTP concentrations iii) Extension of primers with mismatched 3’-OH ends opposite m 6 G. Only m6G:dAMP and m6G:dCMP mismatches were extended by DNA polymerases α, δ and ε from both sources. No differences in extension efficiency were observed between the enzymes from normal and hepatoma cells. Taken together, our results suggest that DNA polymerases α, δ, and ε from Novikoff cells catalyzed incorporation of the wrong nucleotides more readily and extended mismatches more easily. These results may provide a rationale why numerous mutations accumulate during tumor development.

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