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 sy nthesis 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 mali gnant Novikoff hepatoma cells and regenerating normal rat liver and compare d physicochemical and catalytic properties of the DNA polymerase alpha -pri mase complexes purified from both sources. Sequence analysis showed two mut ations in subunit B from Novikoff cells: one in nucleotide position 855 (CC G --> CCA) that did not result in an amino acid exchange and one in positio n 862 (GTG --> ATG) that caused a change of valine to methionine in codon 2 88. No mutation was found in the three other subunits. The wild-type and mu tated sequences of subunit B were cloned and expressed in vitro. Sedimentat ion analysis of the expressed polypeptides revealed different sedimentation constants, indicating that the amino acid exchange affected the conformati on of subunit B. The analysis of the purified DNA polymerase alpha -primase complexes showed a sedimentation value that was significantly higher for t he enzyme complex from normal liver than for that from Novikoff cells. In a ddition, DNA polymerase alpha -primase complexes from Novikoff cells showed higher sensitivity to camptothecin, topotecan, and structurally, related c ompounds (such as (R,S)-7-ethyl-10-hydroxycamptothecin, 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 conformatio nal change of the DNA polymerase alpha -primase complex from Novikoff hepat oma cells. Whether this mutation influences genetic instability or tumor de velopment needs to be explored. (C) 2001 Wiley-Liss, Inc.