Two immunologically distinct human DNA polymerase alpha-primase subpopulations are involved in cellular DNA replication.
Metabolic labeling of primate cells revealed the existence of phosphorylated and hypophosphorylated DNA polymerase alpha-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.
Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, D-20251 Hamburg, Germany.
S
S
Dehde
G
G
Rohaly
O
O
Schub
H P
HP
Nasheuer
W
W
Bohn
J
J
Chemnitz
W
W
Deppert
I
I
Dornreiter
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002460:Cell Line; D019915:DNA Primase / Q000235:genetics* / Q000276:immunology; D004261:DNA Replication; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000276:immunology; D018613:Microscopy, Confocal; D010766:Phosphorylation
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D019915:DNA Primase