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.
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.
Eoff
Robert L
RL
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Irimia
Adriana
A
Angel
Karen C
KC
Egli
Martin
M
Guengerich
F Peter
FP
D000080242:8-Hydroxy-2'-Deoxyguanosine; D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000235:genetics; D019641:DNA, Archaeal / Q000737:chemistry* / Q000235:genetics; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry; D006860:Hydrogen Bonding; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary / Q000235:genetics; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D000080242:8-Hydroxy-2'-Deoxyguanosine; D019951:DNA Polymerase beta; D003849:Deoxyguanosine