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, Marietta Y.W.T. Lee

Research output: Contribution to journalArticlepeer-review

26 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)9561-9569
Number of pages9
JournalJournal of Biological Chemistry
Volume273
Issue number16
DOIs
StatePublished - Apr 17 1998

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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