Archaeoglobus Fulgidus DNA Polymerase D: A Zinc-Binding Protein Inhibited by Hypoxanthine and Uracil. Issue 14 (17th July 2016)

• Record Type: Journal Article
• Authors: Abellón-Ruiz, Javier; Waldron, Kevin J.; Connolly, Bernard A.
• Is Part Of: Journal of molecular biology. Volume 428:Issue 14(2016:Jul. 15)
• Page Start: 2805
• Page End: 2813
• Publication Date: 2016-07-17
• Subjects: Pol-B family-B DNA polymerase -- Pol-D family-D DNA polymerase -- DP1 small (proofreading exonuclease) subunit of DNA polymerase D -- DP2 large (polymerase) subunit of DNA polymerase D -- Afu Archaeoglobus fulgidus -- Pfu Pyrococcus furiosus -- ICP-MS inductively coupled plasma mass spectroscopy; Archaea -- DNA polymerase D -- uracil -- hypoxanthine -- Zn-binding protein; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2016.06.008
• Languages: English
• ISSNs: 0022-2836

Abstract

Abstract: 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. Graphical Abstract: Highlights: Archaeal DNA Polymerase-D binds Zn, this is essential for activity. Archaeal DNA Polymerase-D is inhibited by uracil and hypoxanthine. Inhibition by deaminated bases occurs both in cis and in trans. Deaminated bases are sensed by the large (polymerase) subunit. Polymerase-D has high fidelity, compatible with genome replication.