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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 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.

* 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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