Expression and characterization of the small subunit of human DNA polymerase δ

Yubo Sun, Yunquan Jiang, Peng Zhang, Shan Jian Zhang, Yi Zhou, Bao Qing Li, N. Lan Toomey, Marietta Y.W.T. Lee

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25 Scopus citations

Abstract

DNA polymerase δ is a heterodimer consisting of a catalytic subunit of 125 kDa and a small subunit of 50 kDa (p50). We have overexpressed p50 in Escherichia coli and have characterized the recombinant protein. p50 was readily overexpressed using the pET vector as an insoluble protein. A procedure was developed for its purification and renaturation. Examination of the physicochemical properties of renatured p50 showed that it is a monomeric protein with an apparent molecular weight of 60,000, a Stokes radius of 34 Å, and a sedimentation coefficient of 4.1 S. Its physical properties were indistinguishable from p50 expressed as a soluble protein using the pTACTAC vector. Examination of the effects of recombinant p50 on the activity of DNA polymerase δ showed that p50 is able to slightly stimulate (about 2-fold) the activity of the recombinant 125-kDa catalytic subunit using poly(dA)·oligo(dT) as a template in the absence of proliferating cell nuclear antigen. In the presence of proliferating cell nuclear antigen, activity is stimulated about 5-fold. Seven stable hybridoma cell lines were established that produced monoclonal antibodies against p50. One of these antibodies (13D5) inhibited the activity of calf thymus DNA polymerase δ. This antibody, when coupled to a solid support, also was found to provide a method for the immunoaffinity purification of recombinant p50 and of DNA polymerase δ from calf thymus or HeLa extracts. Immunoprecipitation and enzyme-linked immunosorbent assays also confirmed that p50 interacts with the catalytic subunit of DNA polymerase δ.

Original languageEnglish (US)
Pages (from-to)13013-13018
Number of pages6
JournalJournal of Biological Chemistry
Volume272
Issue number20
DOIs
StatePublished - May 16 1997

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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