Hydrogen bonding of 7,8-dihydro-8-oxodeoxyguanosine with a charged residue in the little finger domain determines miscoding events in Sulfolobus solfataricus DNA polymerase Dpo4

J Biol Chem. 2007 Jul 6;282(27):19831-43. doi: 10.1074/jbc.M702290200. Epub 2007 Apr 27.


Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) has been shown to catalyze bypass of 7,8-dihydro-8-oxodeoxyguanosine (8-oxoG) in a highly efficient and relatively accurate manner. Crystal structures have revealed a potential role for Arg(332) in stabilizing the anti conformation of the 8-oxoG template base by means of a hydrogen bond or ion-dipole pair, which results in an increased enzymatic efficiency for dCTP insertion and makes formation of a Hoogsteen pair between 8-oxoG and dATP less favorable. Site-directed mutagenesis was used to replace Arg(332) with Ala, Glu, Leu, or His in order to probe the importance of Arg(332) in accurate and efficient bypass of 8-oxoG. The double mutant Ala(331)Ala(332) was also prepared to address the contribution of Arg(331). Transientstate kinetic results suggest that Glu(332) retains fidelity against bypass of 8-oxoG that is similar to wild type Dpo4, a result that was confirmed by tandem mass spectrometric analysis of full-length extension products. A crystal structure of the Dpo4 Glu(332) mutant and 8-oxoG:C pair revealed water-mediated hydrogen bonds between Glu(332) and the O-8 atom of 8-oxoG. The space normally occupied by Arg(332) side chain is empty in the crystal structures of the Ala(332) mutant. Two other crystal structures show that a Hoogsteen base pair is formed between 8-oxoG and A in the active site of both Glu(332) and Ala(332) mutants. These results support the view that a bond between Arg(332) and 8-oxoG plays a role in determining the fidelity and efficiency of Dpo4-catalyzed bypass of the lesion.

Publication types

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

MeSH terms

  • 8-Hydroxy-2'-Deoxyguanosine
  • Amino Acid Substitution
  • Archaeal Proteins / chemistry*
  • Archaeal Proteins / metabolism
  • Crystallography, X-Ray
  • DNA Polymerase beta / chemistry*
  • DNA Polymerase beta / genetics
  • DNA, Archaeal / chemistry*
  • DNA, Archaeal / genetics
  • Deoxyguanosine / analogs & derivatives*
  • Deoxyguanosine / chemistry
  • Hydrogen Bonding
  • Mutation, Missense
  • Protein Structure, Tertiary / genetics
  • Sulfolobus solfataricus / enzymology*
  • Sulfolobus solfataricus / genetics


  • Archaeal Proteins
  • DNA, Archaeal
  • 8-Hydroxy-2'-Deoxyguanosine
  • DNA Polymerase beta
  • Deoxyguanosine

Associated data

  • PDB/2UVR
  • PDB/2UVU
  • PDB/2UVV
  • PDB/2UVW