Characterization of the p125 subunit of human DNA polymerase and its deletion mutants: Interaction with cyclin-dependent kinase-cyclins

The catalytic subunit of human DNA polymerase (pol) δ 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 δ 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 δ and cyclin-dependent kinase (cdk)-cyclins followed by metabolic labeling with ³²P(i) showed that the recombinant catalytic subunit of pol δ could be hyperphosphorylated by G₁ phase-specific cdk-cyclins. When cdk2 was coexpressed with pol δ in Sf9 cells, pol δ was found to coimmunoprecipitate with antibodies against cdk2. Experiments with deletion mutants of pol δ 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 δ by cdk2-cyclins had little or no effect on the specific activity of the enzyme.