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 Zn2+ 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.
Highlights
The biochemical properties of family-D enzymes are generally compatible with DNA replication, and it has been proposed that family-D DNA (Pol-D) copies the lagging strand, while family-B DNA polymerase (Pol-B) is involved in the leading strand synthesis [16,17,18,19]
This has been most thoroughly elucidated for Pol-B, where replication is stalled on encountering uracil or hypoxanthine in template strands [25]
It is assumed that the polymerase response to deaminated bases serves to suppress mutations, perhaps via replication fork regression [30], a feature that may be advantageous for organisms that live at high temperatures, which promote cytosine to uracil deamination
Summary
[2,8,9,10,11,12]. in the Crenarchaea, the family-B polymerase is the only enzyme present with features compatible with DNA replication [2,11,12]. Spaced cysteines have been observed in Eukaryotic family-B DNA polymerases and shown to serve as ligands for Zn2+ and an Fe–S cluster [33,34]. (b) Extension of the control (thymidine-containing) primer-template by wild-type Afu Pol-D and mut2, mut4, or mut5 (Fig. 1) for the times indicated in seconds (′′) or minutes (′).
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