Calcium-dependent calmodulin-binding proteins associated with mammalian DNA polymerases α

Abstract

Complex, multiprotein forms of bovine (calf thymus), hamster (Chinese hamster ovary cell), and human (HeLa) cell DNA polymerase α (Polα) were analyzed for their content of calmodulin-binding proteins. The approach used an established autoradiographic technique employing 125I-labeled calmodulin to probe proteins in denaturing SDS-polyacrylamide gel electropherograms. All three Polα enzymes were associated with discrete, Ca 2+-dependent calmodulin-binding proteins. Conventionally purified calf thymus Polα holoenzyme contained three prominent, trifluoperazine-sensitive species with apparent molecular masses of approx. 120, 80 and 48 kDa. The 120 and 48 kDa species remained associated with the polymerase · primase core of the calf enzyme during immunopurification with monoclonal antibodies directed specifically against the polymerase subunit. The patterns of the calmodulin-binding proteins displayed by conventionally purified preparations of hamster and human Polα enzymes were similar to each other and distinctly different from the pattern of comparable preparations of calf thymus Polα. Immunopurified preparations of the human and hamster Polαs retained significant calmodulin-binding activity of apparent molecular masses of approx. 55, 80 and 150–200 kDa.