Dataset Information

Archaeoglobus Fulgidus DNA Polymerase D: A Zinc-Binding Protein Inhibited by Hypoxanthine and Uracil.

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.

SUBMITTER: Abellon-Ruiz J

PROVIDER: S-EPMC4942837 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Archaeoglobus Fulgidus DNA Polymerase D: A Zinc-Binding Protein Inhibited by Hypoxanthine and Uracil.

Abellón-Ruiz Javier J Waldron Kevin J KJ Connolly Bernard A BA

Journal of molecular biology 20160616 14

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 sho ...[more]

PMID: 27320386

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Project description:The family 4 uracil-DNA glycosylase from the hyperthermophilic organism Archaeoglobus fulgidus (AFUDG) is responsible for the removal of uracil in DNA as the first step in the base excision repair (BER) pathway. AFUDG contains a large solvent-exposed peptide region containing an α helix and loop anchored on each end via ligation of two cysteine thiolates to a [4Fe-4S](2+) cluster. We propose that this region plays a similar role in DNA damage recognition as a smaller iron-sulfur cluster loop (FCL) motif in the structurally unrelated BER glycosylases MutY and Endonuclease III and therefore refer to this region as the "pseudo-FCL" in AFUDG. In order to evaluate the importance of this region, three positively charged residues (Arg 86, Arg 91, Lys 100) and the anchoring Cys residues (Cys 85, Cys 101) within this motif were replaced with alanine, and the effects of these replacements on uracil excision in single- and double-stranded DNA were evaluated. These results show that this region participates and allows for efficient recognition and excision of uracil within DNA. Notably, R86A AFUDG exhibited reduced activity for uracil removal only within double-stranded DNA, suggesting an importance in duplex disruption and extrusion of the base as part of the excision process. In addition, mutation of the [4Fe-4S](2+) cluster cysteine ligands at the ends of the pseudo-FCL to alanine reduced the uracil excision efficiency, suggesting the importance of anchoring the loop via coordination to the cluster. In contrast, K100A AFUDG exhibited enhanced uracil excision activity, providing evidence for the importance of the loop conformation and flexibility. Taken together, the results herein provide evidence that the pseudo-FCL motif is involved in DNA binding and catalysis, particularly in duplex DNA contexts. This work underscores the requirement of an ensemble of interactions, both distant and in proximity to the damaged site, for accurate and efficient uracil excision.

Dataset Information

Archaeoglobus Fulgidus DNA Polymerase D: A Zinc-Binding Protein Inhibited by Hypoxanthine and Uracil.

Publications

Archaeoglobus Fulgidus DNA Polymerase D: A Zinc-Binding Protein Inhibited by Hypoxanthine and Uracil.

Similar Datasets

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