Characterization of the p125 Subunit of Human DNA Polymerase δ 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,Marietta Y.W.T Lee

doi:10.1074/jbc.273.16.9561

Sheng-Ming Wu, Peng Zhang + Show 5 more

Open Access

https://doi.org/10.1074/jbc.273.16.9561

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Abstract

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

Highlights

The catalytic subunit of human DNA polymerase ␦ was overexpressed in an active, soluble form by the use of a baculovirus system in insect cells
Characterization of Recombinant p125—The enzymatic properties of the recombinant pol ␦ catalytic subunit were compared with those of native calf thymus pol ␦, which had been isolated by immunoaffinity chromatography [14], and with the endogenous DNA polymerase activity from Sf9 cells infected with wild type Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) (Fig. 8)
The studies reported here show that the catalytic subunit of DNA pol ␦ can be expressed in Sf9 cells in an active form and can be isolated by a conventional purification protocol or by an immunoaffinity chromatography procedure

Summary

EXPERIMENTAL PROCEDURES

Materials—Sf9 cells were purchased from Invitrogen and were maintained at 27 °C in TNM-FH insect medium supplemented with 10% fetal calf serum and 50 ␮g/ml gentamycin. Extracts of Sf9 cells prepared as described above were subjected to SDS-PAGE in 5–15% gradient gels that were transferred to nitrocellulose membranes. The cells were sonicated for 30 s in 40 mM Tris-HCl, pH 7.8, 0.25 M sucrose, 0.5 M NaCl, 0.1% Nonidet P-40, 0.1 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, and 10 mM benzamidine-HCl. The crude cell extracts were transferred to microtubes and centrifuged at 15,000 ϫ g for 30 min. Immunoprecipitation and immunoblotting of Molt 4 Cells with Pol ␦ and Members of the Cyclin and Cdk—4 ϫ 107 exponentially growing Molt 4 cells were prepared and lysed with 300 ␮l of Nonidet P-40 buffer (50 mM Tris-HCl, 1 mM phenylmethylsulfonyl fluoride, 150 mM NaCl, and 1% Nonidet P-40). After SDS-PAGE, the separated proteins were transferred to a nitrocellulose membrane and Western blotted with antibodies to cdk, cdk, or pol ␦

RESULTS

Protein Activity Specific activity Recovery

DISCUSSION