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J Biol Chem. 2001 Oct 12;276(41):38076-83. Epub 2001 Aug 13.

Multiple phosphorylation sites of DNA polymerase alpha-primase cooperate to regulate the initiation of DNA replication in vitro.

Author information

1
Institut für Molekulare Biotechnologie, Abteilung Biochemie, Beutenbergstrasse 11, D-07745 Jena, Germany.

Abstract

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.

PMID:
11502743
DOI:
10.1074/jbc.M104975200
[Indexed for MEDLINE]
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