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J Biol Chem, Vol. 273, Issue 16, 9561-9569, April 17, 1998

Characterization of the p125 Subunit of Human DNA Polymerase  and Its Deletion Mutants
INTERACTION WITH CYCLIN-DEPENDENT KINASE-CYCLINS

Sheng-Ming Wu, Peng Zhang^§, Xiao Rong Zeng, Shan-Jian Zhang, Jinyao Mo^§, Bao Qing Li^§, and Marietta Y. W. T. Lee^§

From the  Department of Biochemistry and Molecular Biology, University of Miami, Miami, Florida 33101 and the ^§ Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595

The catalytic subunit of human DNA polymerase (pol)  was overexpressed in an active, soluble form by the use of a baculovirus system in insect cells. The recombinant enzyme was separated from endogenous DNA polymerases by phosphocellulose, Mono Q-Sepharose, and single-stranded DNA-cellulose chromatography. Recombinant DNA pol  was also purified by immunoaffinity chromatography. The enzymatic properties of the purified catalytic subunit were characterized. The enzyme was active and possessed both DNA polymerase and associated 3' to 5' exonuclease activities. NH₂-terminal deletion mutants retained polymerase activity, whereas the core and COOH-terminal deletion mutants were devoid of any measurable activities. Coinfection of Sf9 cells with recombinant baculovirus vectors for pol  and cyclin-dependent kinase (cdk)-cyclins followed by metabolic labeling with ³²P_i showed that the recombinant catalytic subunit of pol  could be hyperphosphorylated by G₁ phase-specific cdk-cyclins. When cdk2 was coexpressed with pol  in Sf9 cells, pol  was found to coimmunoprecipitate with antibodies against cdk2. Experiments with deletion mutants of pol  showed that the NH₂-terminal region was essential for this interaction. Coimmunoprecipitation and Western blot experiments in Molt 4 cells confirmed the interaction in vivo. Preliminary experiments showed that phosphorylation of the catalytic subunit of pol  by cdk2-cyclins had little or no effect on the specific activity of the enzyme.

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