A mutation in subunit B of the DNA polymerase alpha-primase complex from Novikoff hepatoma cells concomitant with a conformational change and abnormal catalytic properties of the DNA polymerase alpha-primase complex.

Mutated constituents of the DNA replication complex might contribute to the mutational load of the genome during tumor development by impairing DNA synthesis as well as cell cycle-related control of DNA replication. To prove or disprove this hypothesis, we looked for mutations in the cDNA sequences of the four subunits of DNA polymerase alpha-primase from both highly malignant Novikoff hepatoma cells and regenerating normal rat liver and compared physicochemical and catalytic properties of the DNA polymerase alpha-primase complexes purified from both sources. Sequence analysis showed two mutations in subunit B from Novikoff cells: one in nucleotide position 855 (CCG-->CCA) that did not result in an amino acid exchange and one in position 862 (GTG-->ATG) that caused a change of valine to methionine in codon 288. No mutation was found in the three other subunits. The wild-type and mutated sequences of subunit B were cloned and expressed in vitro. Sedimentation analysis of the expressed polypeptides revealed different sedimentation constants, indicating that the amino acid exchange affected the conformation of subunit B. The analysis of the purified DNA polymerase alpha-primase complexes showed a sedimentation value that was significantly higher for the enzyme complex from normal liver than for that from Novikoff cells. In addition, DNA polymerase alpha-primase complexes from Novikoff cells showed higher sensitivity to camptothecin, topotecan, and structurally related compounds (such as (R,S)-7-ethyl-10-hydroxy camptothecin, 9-aminocamptothecin, and 10-hydroxycamptothecin) than the enzyme from normal rat liver. Thus, the amino acid change found in subunit B appears to result in a conformational change of the DNA polymerase alpha-primase complex from Novikoff hepatoma cells. Whether this mutation influences genetic instability or tumor development needs to be explored.