Archaeoglobus Fulgidus DNA Polymerase D: A Zinc-Binding Protein Inhibited by Hypoxanthine and Uracil.
Archaeal family-D DNA polymerases (Pol-D) comprise a small (DP1) proofreading subunit and a large (DP2) polymerase subunit. Pol-D is one of the least studied polymerase families, and this publication investigates the enzyme from Archaeoglobus fulgidus (Afu Pol-D). The C-terminal region of DP2 contains two conserved cysteine clusters, and their roles are investigated using site-directed mutagenesis. The cluster nearest the C terminus is essential for polymerase activity, and the cysteines are shown to serve as ligands for a single, critical Zn(2+) ion. The cysteines farthest from the C terminal were not required for activity, and a role for these amino acids has yet to be defined. Additionally, it is shown that Afu Pol-D activity is slowed by the template strand hypoxanthine, extending previous results that demonstrated inhibition by uracil. Hypoxanthine was a weaker inhibitor than uracil. Investigations with isolated DP2, which has a measurable polymerase activity, localised the deaminated base binding site to this subunit. Uracil and hypoxanthine slowed Afu Pol-D "in trans", that is, a copied DNA strand could be inhibited by a deaminated base in the alternate strand of a replication fork. The error rate of Afu Pol-D, measured in vitro, was 0.24×10(-5), typical for a polymerase that has been proposed to carry out genome replication in the Archaea. Deleting the 3'-5' proofreading exonuclease activity reduced fidelity twofold. The results presented in this publication considerably increase our knowledge of Pol-D.
Institute for Cell and Molecular Biology, University of Newcastle, Newcastle upon Tyne NE2 4HH, UK.
Javier
J
Abellón-Ruiz
Institute for Cell and Molecular Biology, University of Newcastle, Newcastle upon Tyne NE2 4HH, UK.
Kevin J
KJ
Waldron
Institute for Cell and Molecular Biology, University of Newcastle, Newcastle upon Tyne NE2 4HH, UK. Electronic address: bernard.connolly@ncl.ac.uk.
Bernard A
BA
Connolly
D019843:Archaeal Proteins / Q000037:antagonists & inhibitors*; D019611:Archaeoglobus fulgidus / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000037:antagonists & inhibitors*; D003545:Cysteine / Q000235:genetics; D004258:DNA Polymerase III / Q000037:antagonists & inhibitors*; D004261:DNA Replication / Q000235:genetics; D019641:DNA, Archaeal / Q000235:genetics; D019271:Hypoxanthine / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed / Q000379:methods; D014498:Uracil / Q000494:pharmacology*; D015032:Zinc / Q000378:metabolism*
D019843:Archaeal Proteins; D002352:Carrier Proteins; D019641:DNA, Archaeal; C017800:zinc-binding protein; D019271:Hypoxanthine; D014498:Uracil; D004258:DNA Polymerase III; D015032:Zinc; D003545:Cysteine
Archaea; DNA polymerase D; Zn-binding protein; hypoxanthine; uracil