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Molecular and Cellular Biology, April 2001, p. 2581-2593, Vol. 21, No. 7
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.7.2581-2593.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Silke Dehde,1 Gabor Rohaly,1 Oliver Schub,2 Heinz-Peter Nasheuer,2 Wolfgang Bohn,1 Jan Chemnitz,1 Wolfgang Deppert,1 and Irena Dornreiter*,1

Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, D-20251 Hamburg,1 and Institut für Molekulare Biotechnologie, Abteilung Biochemie, D-07745 Jena,2 Germany

Received 31 August 2000/Returned for modification 3 October 2000/Accepted 27 December 2000

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.

* Corresponding author. Mailing address: Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie, Martinistr. 52, D-20251 Hamburg, Germany. Phone: 40-48051-231. Fax: 40-48051-117. E-mail: dornreit{at}hpi.uni-hamburg.de.

Molecular and Cellular Biology, April 2001, p. 2581-2593, Vol. 21, No. 7
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.7.2581-2593.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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