Uracil Recognition in Archaeal DNA Polymerases Captured by X-ray Crystallography

Archaeal family B DNA polymerases bind tightly to template-strand uracil and stall replication on encountering the pro-mutagenic base. This article describes an X-ray crystal structure, at 2.8 Å resolution, of Thermococcus gorgonarius polymerase in complex with a DNA primer–template containing uraci...

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Bibliographic Details
Published in:Journal of molecular biology 2008-09, Vol.381 (3), p.529-539
Main Authors: Firbank, Susan J., Wardle, Josephine, Heslop, Pauline, Lewis, Richard J., Connolly, Bernard A.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
archaea
Archaeal Proteins - chemistry
Archaeal Proteins - metabolism
Binding Sites
Crystallography, X-Ray
DNA polymerase
DNA replication and repair
DNA-Directed DNA Polymerase - chemistry
DNA-Directed DNA Polymerase - metabolism
Hydrogen Bonding
Models, Molecular
Molecular Sequence Data
Oligonucleotides - chemistry
Oligonucleotides - metabolism
Templates, Genetic
Thermococcus - enzymology
Thermococcus gorgonarius
uracil
Uracil - chemistry
Uracil - metabolism
X-ray crystallography
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Summary:Archaeal family B DNA polymerases bind tightly to template-strand uracil and stall replication on encountering the pro-mutagenic base. This article describes an X-ray crystal structure, at 2.8 Å resolution, of Thermococcus gorgonarius polymerase in complex with a DNA primer–template containing uracil in the single-stranded region. The DNA backbone is distorted to position the uracil deeply within a pocket, located in the amino-terminal domain of the polymerase. Specificity arises from a combination of hydrogen bonds between the protein backbone and uracil, with the pocket shaped to prevent the stable binding of the four standard DNA bases. Strong interactions are seen with the two phosphates that flank the uracil and the structure gives clues concerning the coupling of uracil binding to the halting of replication. The importance of key amino acids, identified by the analysis of the structure and their conservation between archaeal polymerases, was confirmed by site-directed mutagenesis. The crystal structure of V93Q, a polymerase variant that no longer recognises uracil, is also reported, explaining the V93Q phenotype by the steric exclusion of uracil from the pocket.
ISSN:0022-2836
1089-8638
DOI:10.1016/j.jmb.2008.06.004