Structural and functional elucidation of the mechanism promoting error-prone synthesis by human DNA polymerase kappa opposite the 7,8-dihydro-8-oxo-2'-deoxyguanosine adduct

J Biol Chem. 2009 Aug 14;284(33):22467-22480. doi: 10.1074/jbc.M109.003905. Epub 2009 Jun 19.


Human polymerase kappa (hPol kappa) is one of four eukaryotic Y-class DNA polymerases and may be an important element in the cellular response to polycyclic aromatic hydrocarbons such as benzo[a]pyrene, which can lead to reactive oxygenated metabolite-mediated oxidative stress. Here, we present a detailed analysis of the activity and specificity of hPol kappa bypass opposite the major oxidative adduct 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxoG). Unlike its archaeal homolog Dpo4, hPol kappa bypasses this lesion in an error-prone fashion by inserting mainly dATP. Analysis of transient-state kinetics shows diminished "bursts" for dATP:8-oxoG and dCTP:8-oxoG incorporation, indicative of non-productive complex formation, but dATP:8-oxoG insertion events that do occur are 2-fold more efficient than dCTP:G insertion events. Crystal structures of ternary hPol kappa complexes with adducted template-primer DNA reveal non-productive (dGTP and dATP) alignments of incoming nucleotide and 8-oxoG. Structural limitations placed upon the hPol kappa by interactions between the N-clasp and finger domains combined with stabilization of the syn-oriented template 8-oxoG through the side chain of Met-135 both appear to contribute to error-prone bypass. Mutating Leu-508 in the little finger domain of hPol kappa to lysine modulates the insertion opposite 8-oxoG toward more accurate bypass, similar to previous findings with Dpo4. Our structural and activity data provide insight into important mechanistic aspects of error-prone bypass of 8-oxoG by hPol kappa compared with accurate and efficient bypass of the lesion by Dpo4 and polymerase eta.

Publication types

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

MeSH terms

  • 8-Hydroxy-2'-Deoxyguanosine
  • Base Sequence
  • Catalysis
  • DNA Replication*
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / physiology*
  • Deoxyguanosine / analogs & derivatives*
  • Deoxyguanosine / chemistry
  • Fungal Proteins / metabolism
  • Humans
  • Kinetics
  • Models, Biological
  • Molecular Conformation
  • Molecular Sequence Data
  • Mutation
  • Protein Structure, Tertiary
  • Sulfolobus
  • X-Ray Diffraction


  • Fungal Proteins
  • 8-Hydroxy-2'-Deoxyguanosine
  • DNA-Directed DNA Polymerase
  • POLK protein, human
  • Deoxyguanosine

Associated data

  • PDB/2W7O
  • PDB/2W7P