Cell Cycle-Dependent Regulation of Human DNA Polymerase alpha -Primase Activity by Phosphorylation

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Molecular and Cellular Biology, January 1999, p. 646-656, Vol. 19, No. 1
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Cell Cycle-Dependent Regulation of Human DNA Polymerase $alpha$ -Primase Activity by Phosphorylation

Christian Voitenleitner,¹ Christoph Rehfuess,¹ Melissa Hilmes,¹ Lynda O'Rear,¹ Pao-Chi Liao,² Douglas A. Gage,³ Robert Ott,¹ Heinz-Peter Nasheuer,⁴ and Ellen Fanning¹^,^*

Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, and Vanderbilt Cancer Center, Nashville, Tennessee 37232-6838¹; Department of Environmental and Occupational Health, National Cheng Kung University Medical College, Tainan, 70428 Taiwan, Republic of China²; MSU-NIH Mass Spectrometry Facility and Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824³; and Institut für Molekulare Biotechnologie, Abteilung Biochemie, 07745 Jena, Germany⁴

Received 26 May 1998/Returned for modification 21 July 1998/Accepted 29 September 1998

DNA polymerase $alpha$ -primase is known to be phosphorylated in human and yeast cells in a cell cycle-dependent manner on the p180and p68 subunits. Here we show that phosphorylation of purifiedhuman DNA polymerase $alpha$ -primase by purified cyclin A/cdk2 in vitroreduced its ability to initiate simian virus 40 (SV40) DNA replicationin vitro, while phosphorylation by cyclin E/cdk2 stimulated itsinitiation activity. Tryptic phosphopeptide mapping revealed afamily of p68 peptides that was modified well by cyclin A/cdk2and poorly by cyclin E/cdk2. The p180 phosphopeptides were identicalwith both kinases. By mass spectrometry, the p68 peptide familywas identified as residues 141 to 160. Cyclin A/cdk2- and cyclinA/cdc2-modified p68 also displayed a phosphorylation-dependentshift to slower electrophoretic mobility. Mutation of the fourputative phosphorylation sites within p68 peptide residues 141to 160 prevented its phosphorylation by cyclin A/cdk2 and theinhibition of replication activity. Phosphopeptide maps of thep68 subunit of DNA polymerase $alpha$ -primase from human cells, synchronizedand labeled in G₁/S and in G₂, revealed a cyclin E/cdk2-like patternin G₁/S and a cyclin A/cdk2-like pattern in G₂. The slower-electrophoretic-mobilityform of p68 was absent in human cells in G₁/S and appeared asthe cells entered G₂/M. Consistent with this, the ability of DNApolymerase $alpha$ -primase isolated from synchronized human cells toinitiate SV40 replication was maximal in G₁/S, decreased as thecells completed S phase, and reached a minimum in G₂/M. Theseresults suggest that the replication activity of DNA polymerase $alpha$ -primase in human cells is regulated by phosphorylation in acell cycle-dependentmanner.

^* Corresponding author. Mailing address: Department of Molecular Biology, Box 1820, Station B, Vanderbilt University, Nashville,TN 37235. Phone: (615) 343-5677. Fax: (615) 343-6707. E-mail:FANNINE{at}CTRVAX.VANDERBILT.EDU.

Molecular and Cellular Biology, January 1999, p. 646-656, Vol. 19, No. 1
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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Cell Cycle-Dependent Regulation of Human DNA Polymerase -Primase Activity by Phosphorylation

Cell Cycle-Dependent Regulation of Human DNA Polymerase $alpha$ -Primase Activity by Phosphorylation