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PDBsum entry 2uvr
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References listed in PDB file
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Key reference
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Title
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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.
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Authors
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R.L.Eoff,
A.Irimia,
K.C.Angel,
M.Egli,
F.P.Guengerich.
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Ref.
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J Biol Chem, 2007,
282,
19831-19843.
[DOI no: ]
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PubMed id
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Abstract
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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.
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Figure 5.
Hydrogen bonding interactions between Dpo4 and the 8-oxoG
lesion.A, R332A(8-oxoG:A); B, R332A(8-oxoG:C); C,
R332E(8-oxoG:A); D, R332E(8-oxoG:C); E, wild type 8-oxoG:dATP;
F, wild type 8-oxoG:dCTP. The protein is shown with schematic
α-helices and β-strands. The DNA duplex and selected Dpo4
residues are shown in stick mode. Ca^2+ ions and water molecules
are shown as yellow and red spheres, respectively, and hydrogen
bonds as dashed lines.
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Figure 7.
Superposition of the little finger domains of wild type Dpo4
andS. cerevisiae Pol η. Dpo4 and Pol η (Protein Data Bank
accession code 1jih (44)) are represented schematically with
secondary structure elements colored blue and red, respectively.
The positions of the Cα atoms of Arg^332 (Dpo4) and Lys^498
(Pol η) are shown as spheres.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2007,
282,
19831-19843)
copyright 2007.
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