DNA polymerase alpha from normal rat liver is different than DNA polymerases alpha from Morris hepatoma strains

To investigate whether DNA replication in rat hepatoma cells is altered compared with that in normal rat liver, the main replicative enzyme, i.e. the DNA polymerase alpha complex, was partially purified from a slow-growing (TC5123) and a fast-growing (MH3924) Morris hepatoma cell strain as well as from normal rat liver. The purified DNA polymerase alpha complexes contained RNA primase. DNA polymerase alpha activities of these complexes were characterized with regard to both their molecular properties and their dNTP and DNA binding sites. The latter were probed with competitive inhibitors of dNTP binding, resulting in Ki values, and with DNA templates, yielding Km values. The sedimentation coefficients of native DNA polymerases alpha from Morris hepatoma cells were found to be lower than that of polymerase alpha from normal rat liver. Consequently, when following the procedure of Siegel and Monty for determination of molecular mass considerably smaller molecular masses were calculated for polymerases of hepatoma strains (TC5123, 127 kDa; MH3924, 138 kDa; rat liver, 168 kDa). Similar differences were found when the dNTP binding site was probed with inhibitors. Ki values obtained with butylphenyl-dGTP were higher for polymerases of the hepatoma strains than for that of normal rat liver. However, Ki values measured with aphidicolin and butylanilino-dATP were lower for DNA polymerase alpha from the fast-growing hepatoma cell strain than for that from normal rat liver, indicating a reduced affinity of the dNTP binding sites for dATP and dCTP. This reduced affinity could be responsible for lowered specificity of nucleotide selection in the base-pairing process which in turn may cause an enhanced error rate in DNA replication in malignant cells. Furthermore, when the DNA binding site was characterized by Michaelis-Menten constants using gapped DNA as a template, Km values were similar for all three DNA polymerases. In contrast, the Km value measured with single-stranded DNA as a template was found to be lower for DNA polymerase alpha from the fast-growing hepatoma MH3924 than for that from normal rat liver. Thus, the DNA-polymerizing complex from MH3924 combines both higher binding strength to single-stranded DNA templates and decreased nucleotide selection, properties which may enhance replication velocity and may lower fidelity.