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 Arg332 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 Arg332 with Ala, Glu, Leu, or His in order to probe the importance of Arg332 in accurate and efficient bypass of 8-oxoG. The double mutant Ala331 Ala 332 was also prepared to address the contribution of Arg 331. Transient-state kinetic results suggest that Glu332 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 Glu332 mutant and 8-oxoG:C pair revealed water-mediated hydrogen bonds between Glu332 and the O-8 atom of 8-oxoG. The space normally occupied by Arg332 side chain is empty in the crystal structures of the Ala332 mutant. Two other crystal structures show that a Hoogsteen base pair is formed between 8-oxoG and A in the active site of both Glu332 and Ala332 mutants. These results support the view that a bond between Arg332 and 8-oxoG plays a role in determining the fidelity and efficiency of Dpo4-catalyzed bypass of the lesion. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.