Protein affinity chromatography reveals cell cycle dependent association of cellular factors with human DNA...

DNA polymerase alpha/primase (Pol alpha) is the key replication enzyme in eukaryotic cells. This enzyme synthesizes and elongates short RNA primers at an unwound origin of replication. Pol alpha was used as an affinity ligand to identify cellular replication factors interacting with it. Protein complexes between Pol alpha and cellular factors were analyzed by co-immunoprecipitations with monoclonal antibodies directed against Pol alpha and by protein affinity chromatography of cell extracts derived from pure G1- and S-phase cell populations on Pol alpha affinity columns. Co-immunoprecipitations resulted in the identification of a polypeptide with a molecular weight of 46 kDa. For Pol alpha affinity chromatography, the ligand was purified from insect cells infected with a recombinant baculovirus encoding the catalytic subunit (p180) of Pol alpha (Copeland and Wang, 1991). With 5 x 10(8) infected Sf9 cells, a rapid one step purification protocol was used which yielded in five hours 0.6 mg pure enzyme with a specific activity of 140,000 units/mg. The G1- and S-phase cell populations were generated by block, release and counterflow centrifugal elutriation of exponentially growing human MANCA cells. Starting with 2 x 10(9) non synchronous cells, 5 x 10(8) G1-phase cells were isolated. Chromatography of cell extracts derived from G1- or S-phase cells on Pol alpha affinity columns resulted in identifying several polypeptides in the range of 40-70 kDa. Some of these polypeptides are more abundant in eluates derived from S-phase extracts than from G1-phase extracts.