DNA polymerases are essential enzymes in all domains of life for both DNA replication and repair. The primary DNA replication polymerase from Sulfolobus solfataricus (SsoDpo1) has been shown previously to provide the necessary polymerization speed and exonuclease activity to replicate the genome accurately. We find that this polymerase is able to physically associate with itself to form a trimer and that this complex is stabilized in the presence of DNA. Analytical gel filtration and electrophoretic mobility shift assays establish that initially a single DNA polymerase binds to DNA followed by the cooperative binding of two additional molecules of the polymerase at higher concentrations of the enzyme. Protein chemical crosslinking experiments show that these are specific polymerase–polymerase interactions and not just separate binding events along DNA. Isothermal titration calorimetry and fluorescence anisotropy experiments corroborate these findings and show a stoichiometry where three polymerases are bound to a single DNA substrate. The trimeric polymerase complex significantly increases both the DNA synthesis rate and the processivity of SsoDpo1. Taken together, these results suggest the presence of a trimeric DNA polymerase complex that is able to synthesize long DNA strands more efficiently than the monomeric form.

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Differentiation of binding accurate DNA replication polymerases over error prone DNA lesion bypass polymerases is essential for the proper maintenance of the genome. The hyperthermophilic archaeal organism, Sulfolobus solfataricus (Sso), contains both a B-family replication (Dpo1) and a Y-family repair (Dpo4) polymerase and serves as a model system for understanding molecular mechanisms and assemblies for DNA replication and repair protein complexes. Protein crosslinking, isothermal titration calorimetry, and analytical ultracentrifugation have confirmed a previously unrecognized dimeric Dpo4 complex bound to DNA. Binding discrimination between these polymerases on model DNA templates is complicated by the fact that multiple oligomeric species are influenced by concentration and temperature. Temperature dependent fluorescence anisotropy equilibrium binding experiments were used to separate discrete binding events for formation of trimeric Dpo1 and dimeric Dpo4 complexes on DNA. The associated equilibria are found to be temperature dependent, generally leading to improved binding at higher temperatures for both polymerases. At high temperatures, DNA binding by Dpo1 monomer is favored over Dpo4 monomer, but binding of Dpo1 trimer is even more strongly favored over Dpo4 dimer, thus providing thermodynamic selection. Greater processivities of nucleotide incorporation for trimeric Dpo1 and dimeric Dpo4 are also observed at higher temperatures, providing biochemical validation for the influence of tightly bound oligomeric polymerases. These results separate, quantify, and confirm individual and sequential processes leading to formation of oligomeric Dpo1 and Dpo4 assemblies on DNA and provide for a concentration and temperature dependent discrimination of binding undamaged DNA templates at physiological temperatures.

DNA replication polymerases have the inherent ability to faithfully and rapidly copy a DNA template according to precise Watson-Crick base pairing. The primary B-family DNA replication polymerase (Dpo1) in the hyperthermophilic archaeon, Sulfolobus solfataricus, is shown here to possess a remarkable DNA stabilizing ability for maintaining weak base pairing interactions to facilitate primer extension. This thermal stabilization by Dpo1 allowed for template-directed synthesis at temperatures more than 30 °C above the melting temperature of naked DNA. Surprisingly, Dpo1 also displays a competing terminal deoxynucleotide transferase (TdT) activity unlike any other B-family DNA polymerase. Dpo1 is shown to elongate single-stranded DNA in template-dependent and template-independent manners. Experiments with different homopolymeric templates indicate that initial deoxyribonucleotide incorporation is complementary to the template. Rate-limiting steps that include looping back and annealing to the template allow for a unique template-dependent terminal transferase activity. The multiple activities of this unique B-family DNA polymerase make this enzyme an essential component for DNA replication and DNA repair for the maintenance of the archaeal genome at high temperatures.

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