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

J Biol Chem. 2001 Jan 5;276(1):92-8. doi: 10.1074/jbc.M004413200.


The Escherichia coli dinB gene encodes DNA polymerase (pol) 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 pol kappa. Human pol kappa 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 pol kappa possesses optimal activity at 37 degrees C over the pH range 6.5-7.5, and is insensitive to inhibition by aphidicolin, dideoxynucleotides, or NaCl up to 50 mm. Either Mg(2+) or Mn(2+) can satisfy a metal cofactor requirement for pol kappa activity, with Mg(2+) being preferred. Human pol kappa is unable to bypass a cisplatin adduct in the template. However, pol kappa 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 pol kappa 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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetoxyacetylaminofluorene / metabolism
  • Acetoxyacetylaminofluorene / pharmacology
  • Alkylating Agents / metabolism
  • Alkylating Agents / pharmacology
  • Baculoviridae / genetics
  • Cisplatin / metabolism
  • DNA Adducts / metabolism
  • DNA Damage / drug effects
  • DNA Damage / genetics
  • DNA Polymerase beta / chemistry
  • DNA Polymerase beta / genetics
  • DNA Polymerase beta / isolation & purification*
  • DNA Polymerase beta / metabolism*
  • DNA-Directed DNA Polymerase*
  • Exonucleases / metabolism
  • Humans
  • Hydrogen-Ion Concentration
  • Mutagenesis / drug effects
  • Mutagenesis / genetics
  • Mutation
  • Proliferating Cell Nuclear Antigen / pharmacology
  • Proteins / chemistry
  • Proteins / genetics
  • Proteins / isolation & purification*
  • Proteins / metabolism*
  • Recombinant Fusion Proteins / metabolism
  • Templates, Genetic


  • Alkylating Agents
  • DNA Adducts
  • Proliferating Cell Nuclear Antigen
  • Proteins
  • Recombinant Fusion Proteins
  • cisplatin-DNA adduct
  • Acetoxyacetylaminofluorene
  • DNA Polymerase beta
  • DNA-Directed DNA Polymerase
  • POLK protein, human
  • Exonucleases
  • Cisplatin