title: 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.
abstract: 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.
authors.affiliation: Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Eoff
authors.forename: Adriana
authors.initials: A
authors.lastname: Irimia
authors.forename: Karen C
authors.initials: KC
authors.lastname: Angel
authors.forename: Martin
authors.initials: M
authors.lastname: Egli
authors.forename: F Peter
authors.initials: FP
authors.lastname: Guengerich
mesh_terms: 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
chemical_list: D019843:Archaeal Proteins; D019641:DNA, Archaeal; D000080242:8-Hydroxy-2'-Deoxyguanosine; D019951:DNA Polymerase beta; D003849:Deoxyguanosine