2UVR: Crystal Structures Of Mutant Dpo4 Dna Polymerases With 8- Oxog Containing Dna Template-Primer Constructs

Citation:
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.
PDB ID: 2UVRDownload
MMDB ID: 54327
PDB Deposition Date: 2007/3/13
Updated in MMDB: 2012/10
Experimental Method:
x-ray diffraction
Resolution: 2.9  Å
Source Organism:
Sulfolobus solfataricus P2
Similar Structures:
Biological Unit for 2UVR: trimeric; determined by author and by software (PQS)
Molecular Components in 2UVR
Label Count Molecule
Protein (1 molecule)
1
DNA Polymerase IV(Gene symbol: SSO_RS11880)
Molecule annotation
Nucleotides(2 molecules)
1
5'-d(*gp*gp*gp*gp*gp*ap*ap*gp*gp*ap *tp*tp*cp*c)-3'
Molecule annotation
1
5'-d(*tp*cp*ap*cp*8ogp*gp*ap*ap*tp*cp *cp*tp*tp*cp*cp*cp*cp*c)-3'
Molecule annotation
Chemicals (4 molecules)
1
1
2
3
* Click molecule labels to explore molecular sequence information.

Citing MMDB
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