Multiple phosphorylation sites of DNA polymerase alpha-primase cooperate to regulate the initiation of DNA replication in vitro.
DNA polymerase alpha-primase (pol-prim) is the only enzyme that can start DNA replication de novo. The 180-kDa (p180) and 68-kDa (p68) subunits of the human four-subunit enzyme are phosphorylated by Cyclin-dependent kinases (Cdks) in a cell cycle-dependent manner. Cyclin A-Cdk2 physically interacts with pol-prim and phosphorylates N-terminal amino acids of the p180 and the p68 subunits, leading to an inhibition of pol-prim in initiating cell-free SV40 DNA replication. Mutation of conserved putative Cdk phosphorylation sites in the N terminus of human p180 and p68 reduced their phosphorylation by Cyclin A-Cdk2 in vitro. In contrast to wild-type pol-prim these mutants were no longer inhibited by Cyclin A-Cdk2 in the initiation of viral DNA replication. Importantly, rather than inhibiting it, Cyclin A-Cdk2 stimulated the initiation activity of pol-prim containing a triple N-terminal alanine mutant of the p180 subunit. Together these results suggest that Cyclin A-Cdk2 executes both stimulatory and inhibitory effects on the activity of pol-prim in initiating DNA replication.
Schub
O
O
Institut für Molekulare Biotechnologie, Abteilung Biochemie, Beutenbergstrasse 11, D-07745 Jena, Germany.
Rohaly
G
G
Smith
R W
RW
Schneider
A
A
Dehde
S
S
Dornreiter
I
I
Nasheuer
H P
HP
D000818:Animals; D042846:CDC2-CDC28 Kinases; D002478:Cells, Cultured; D019925:Cyclin A / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004256:DNA Polymerase I / Q000737:chemistry / Q000378:metabolism*; D019915:DNA Primase / Q000737:chemistry / Q000378:metabolism*; D004261:DNA Replication; D006801:Humans; D007313:Insecta; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism
D019925:Cyclin A; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D019915:DNA Primase; C116932:DNA polymerase alpha-primase; D004256:DNA Polymerase I