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PMID: 9858588 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

Cell cycle-dependent regulation of human DNA polymerase alpha-primase activity by phosphorylation.

Molecular and cellular biology ·Vol. 19 ·No. 1 ·1999-01-00 ·Pages 646-56

Voitenleitner C, Rehfuess C, Hilmes M, O'Rear L, Liao PC, Gage DA, Ott R, Nasheuer HP, Fanning E

Abstract

DNA polymerase alpha-primase is known to be phosphorylated in human and yeast cells in a cell cycle-dependent manner on the p180 and p68 subunits. Here we show that phosphorylation of purified human DNA polymerase alpha-primase by purified cyclin A/cdk2 in vitro reduced its ability to initiate simian virus 40 (SV40) DNA replication in vitro, while phosphorylation by cyclin E/cdk2 stimulated its initiation activity. Tryptic phosphopeptide mapping revealed a family of p68 peptides that was modified well by cyclin A/cdk2 and poorly by cyclin E/cdk2. The p180 phosphopeptides were identical with both kinases. By mass spectrometry, the p68 peptide family was identified as residues 141 to 160. Cyclin A/cdk2- and cyclin A/cdc2-modified p68 also displayed a phosphorylation-dependent shift to slower electrophoretic mobility. Mutation of the four putative phosphorylation sites within p68 peptide residues 141 to 160 prevented its phosphorylation by cyclin A/cdk2 and the inhibition of replication activity. Phosphopeptide maps of the p68 subunit of DNA polymerase alpha-primase from human cells, synchronized and labeled in G1/S and in G2, revealed a cyclin E/cdk2-like pattern in G1/S and a cyclin A/cdk2-like pattern in G2. The slower-electrophoretic-mobility form of p68 was absent in human cells in G1/S and appeared as the cells entered G2/M. Consistent with this, the ability of DNA polymerase alpha-primase isolated from synchronized human cells to initiate SV40 replication was maximal in G1/S, decreased as the cells completed S phase, and reached a minimum in G2/M. These results suggest that the replication activity of DNA polymerase alpha-primase in human cells is regulated by phosphorylation in a cell cycle-dependent manner.

MeSH Terms
Animals Binding Sites CDC2-CDC28 Kinases Cell Cycle Cell Line Cyclin A/metabolism Cyclin-Dependent Kinase 2 Cyclin-Dependent Kinases/metabolism DNA Polymerase I/metabolism DNA Primase/metabolism DNA Replication Humans Peptide Mapping Phosphopeptides/metabolism Phosphorylation Protein Serine-Threonine Kinases/metabolism Recombinant Fusion Proteins/genetics,metabolism Trypsin
Chemicals
Cyclin A Phosphopeptides Recombinant Fusion Proteins Protein Serine-Threonine Kinases CDC2-CDC28 Kinases CDK2 protein, human Cyclin-Dependent Kinase 2 Cyclin-Dependent Kinases DNA Primase DNA polymerase alpha-primase DNA Polymerase I Trypsin
Authors & Affiliations
9 authors, click to expand affiliations / ORCID
Voitenleitner C
Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, and Vanderbilt Cancer Center, Nashville, Tennessee 37232-6838, USA.
Rehfuess C
Hilmes M
O'Rear L
Liao P C
Gage D A
Ott R
Nasheuer H P
Fanning E
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.19.1.646
Published
1999-01-00
Pages
646-56
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC83922
Subset
IM
Grants
NIGMS NIH HHS · R01 GM 52948 · United States
NCRR NIH HHS · RR00480 · United States
External Links
PubMed source DOI: 10.1128/MCB.19.1.646 PMC Full Text View journal details
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