Differential temperature-dependent multimeric assemblies of replication and repair polymerases on DNA increase processivity.

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 cross-linking, 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 the 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 of Dpo1 monomer is favored over binding of Dpo4 monomer, but binding of Dpo1 trimer is even more strongly favored over binding of 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 the 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.