Probing the Interaction of Archaeal DNA Polymerases with Deaminated Bases Using X-ray Crystallography and Non-Hydrogen Bonding Isosteric Base Analogues

摘要

Archaeal family-B DNA polymerases stall replication on encountering the pro-mutagenic basesnuracil and hypoxanthine. This publication describes an X-ray crystal structure of Thermococcus gorgonariusnpolymerase in complex with a DNA containing hypoxanthine in the single-stranded region of the template,ntwo bases ahead of the primer-template junction. Full details of the specific recognition of hypoxanthine arenrevealed, allowing a comparison with published data that describe uracil binding. The two bases arenrecognized by the same pocket, in the N-terminal domain, and make very similar protein-DNA interactions.nSpecificity for hypoxanthine (and uracil) arises froma combination of polymerase-base hydrogen bonds andnshape fit between the deaminated bases and the pocket. The structurewith hypoxanthine at position 2 explainsnthe stimulation of the polymerase 30n-50nproofreading exonuclease, observed with deaminated bases at thisnlocation. A β-hairpin element, involved in partitioning the primer strand between the polymerase andnexonuclease active sites, inserts between the two template bases at the extreme end of the double-strandednDNA. This denatures the two complementary primer bases and directs the resulting 30nsingle-strandednextension toward the exonuclease active site. Finally, the relative importance of hydrogen bonding and shapenfit in determining selectivity for deaminated bases has been examined using nonpolar isosteres. Affinity fornboth 2,4-difluorobenzene and fluorobenzimidazole, non-hydrogen bonding shape mimics of uracil andnhypoxanthine, respectively, is strongly diminished, suggesting polar protein-base contacts are important.nHowever, residual interaction with 2,4-difluorobenzene is seen, confirming a role for shape recognition.