A mutation detected in DNA polymerase delta cDNA from Novikoff hepatoma cells correlates with abnormal catalytic properties of the enzyme

Tumor development is characterized by accumulation of mutations. Such mutat ions, if induced by carcinogens in DNA polymerase genes, would confer mutat or properties on the DNA replication machinery, even at later stages of dev elopment. To investigate whether DNA polymerase delta can be mutated, we co mpared these enzymes from highly malignant Novikoff hepatoma cells and from regenerating normal rat liver. We sequenced the DNA polymerase delta cDNA from both sources and investigated the physico-chemical properties, inhibit ion characteristics, and copying fidelity of the purified enzymes. The cDNA sequences examined included the entire reading frame encoding the catalyti c subunit (subunit I) of DNA polymerase delta. First-strand cDNAs were prep ared from total RNA of both normal rat liver and Novikoff cells by reverse transcription, and the polymerase delta sequences were amplified by the pol ymerase chain reaction. cDNA (3325 bp) were sequenced. A single heterozygou s mutation (CGG --> CAG) has been detected in nucleotide position 1948 (cod on 648) of the polymerase delta gene from Novikoff cells, resulting in an A rg to Gln change, Position 648 lies just proximal to the conserved region V I, which is part of the "fingers" subdomain of a-like polymerases. This sub domain is involved in dNTP binding. Upon comparison of biochemical characte ristics of partially purified DNA polymerase delta from both Novikoff cells and rat liver, the following properties of the enzyme from Novikoff cells were found to be altered: (i) K-50 values for nucleotide analogs (e.g. buty lphenyl-dGTP) were lower, (ii) sensitivity to various antineoplastic drugs (e.g. doxorubicin, topotecan and distamycin) was enhanced, (iii) copying fi delity was decreased when primer templates containing O-6-methylguanine wer e used, and (iv) the activity of DNA polymerase delta from Novikoff tumor c ells was less stimulated by lactate dehydrogenase than the enzyme from norm al cells. The altered biochemical characteristics of DNA polymerase delta f rom Novikoff cells suggest mutator properties. We conclude that the point m utation detected in the cDNA might be causally related to the observed chan ges in inhibition characteristics and copying fidelity.