2uvu

From Proteopedia

Revision as of 16:33, 5 November 2007

CRYSTAL STRUCTURES OF MUTANT DPO4 DNA POLYMERASES WITH 8-OXOG CONTAINING DNA TEMPLATE-PRIMER CONSTRUCTS

Overview

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.

About this Structure

2UVU is a Single protein structure of sequence from Sulfolobus solfataricus with CA and DGT as ligands. Active as DNA-directed DNA polymerase, with EC number 2.7.7.7 Structure known Active Site: AC1. Full crystallographic information is available from OCA.

Reference

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., Eoff RL, Irimia A, Angel KC, Egli M, Guengerich FP, J Biol Chem. 2007 Jul 6;282(27):19831-43. Epub 2007 Apr 27. PMID:17468100

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