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.