Purification and characterization of polκ, a DNA polymerase encoded by the human DINB1 gene

V. L. Gerlach, W. J. Feaver, P. L. Fischhaber, E. C. Friedberg

Research output: Contribution to journalArticlepeer-review

112 Scopus citations

Abstract

The Escherichia coli dinB gene encodes DNA polymerase (pod IV, a protein involved in increasing spontaneous mutations in vivo. The protein-coding region of DINB1, the human ortholog of DNA pol IV, was fused to glutathione S-transferase and expressed in insect cells. The purified fusion protein was shown to be a template-directed DNA polymerase that we propose to designate polκ. Human polκ lacks detectable 3′ → 5′ proofreading exonuclease activity and is not stimulated by recombinant human proliferating cell nuclear antigen in vitro. Between pH 6.5 and 8.5, human polκ possesses optimal activity at 37 °C over the pit range 6.5-7.5, and is insensitive to inhibition by aphidicolin, dideoxynucleotides, or NaCl up to 50 mM. Either Mg2+ or Mn2+ can satisfy a metal cofactor requirement for polκ activity, with Mg2+ being preferred. Human pole is unable to bypass a cisplatin adduct in the template. However, polκ shows limited bypass of an 2-acetylaminofluorene lesion and can incorporate dCTP or dTTP across from this lesion, suggesting that the bypass is potentially mutagenic. These results are consistent with a model in which pole acts as a specialized DNA polymerase whose possible role is to facilitate the replication of templates containing abnormal bases, or possessing structurally aberrant replication forks that inhibit normal DNA synthesis.

Original languageEnglish (US)
Pages (from-to)92-98
Number of pages7
JournalJournal of Biological Chemistry
Volume276
Issue number1
DOIs
StatePublished - Jan 5 2001

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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