Two Immunologically Distinct Human DNA Polymerase α-Primase Subpopulations Are Involved in Cellular DNA Replication

Abstract

Metabolic labeling of primate cells revealed the existence of phosphorylated and hypophosphorylated DNA polymerase α-primase (Pol-Prim) populations that are distinguishable by monoclonal antibodies. Cell cycle studies showed that the hypophosphorylated form was found in a complex with PP2A and cyclin E-Cdk2 in G₁, whereas the phosphorylated enzyme was associated with a cyclin A kinase in S and G₂. Modification of Pol-Prim by PP2A and Cdks regulated the interaction with the simian virus 40 origin-binding protein large T antigen and thus initiation of DNA replication. Confocal microscopy demonstrated nuclear colocalization of hypophosphorylated Pol-Prim with MCM2 in S phase nuclei, but its presence preceded 5-bromo-2′-deoxyuridine (BrdU) incorporation. The phosphorylated replicase exclusively colocalized with the BrdU signal, but not with MCM2. Immunoprecipitation experiments proved that only hypophosphorylated Pol-Prim associated with MCM2. The data indicate that the hypophosphorylated enzyme initiates DNA replication at origins, and the phosphorylated form synthesizes the primers for the lagging strand of the replication fork.

ACKNOWLEDGMENTS

We are grateful to T. S.-F. Wang for the recombinant baculovirus encoding human Pol α, B. Hemmings for recombinant baculovirus encoding PP2A, S. E. Kearsey for MCM2 antisera, G. Walter for providing the anti-EE hybridoma, and K. Weisshart for purified human RP-A.

The financial support of the Deutsche Forschungsgemeinschaft (De212/8–2, Na190/6–3, Na190/8–1, and Na190/12–1), Deutsche Krebshilfe (10–1417-De), and NATO (CRG920123) is gratefully acknowledged. The IMB is financially supported by Freistaat Thüringen and Bundesministerium für Bildung und Forschung. The Heinrich-Pette-Institute is financially supported by Freie und Hansestadt Hamburg and Bundesministerium für Gesundheit.