Differential Temperature-Dependent Multimeric Assemblies of Replication and Repair Polymerases on DNA Increase Processivity

Abstract

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.

A hand-off of DNA between archaeal polymerases allows high-fidelity replication to resume at a discrete intermediate three bases past 8-oxoguanine

“…6a Scheme iii). This suggests that PolB1 is more stably bound to DNA relative to PolY, which is consistent with previous Pol:DNA binding affinity studies (52), yet these binding affinities have not been evaluated in the presence of PCNA123. The apparent difference in hand-off rates observed in this study indicates that the fundamental basis for each hand-off is molecularly unique, and additional investigation is also required to obtain direct insight into the kinetic and structural mechanisms of these Pol hand-off events.…”

Hybrid Methods Reveal Multiple Flexibly Linked DNA Polymerases within the Bacteriophage T7 Replisome

“…Compared to the bound orientation of Pol A and Pol B on gp4, Pol C is rotated ~90 degrees around an axis that is tipped slightly out of the plane of the gp4 ring (Figure S1). The function of this “extra” copy of DNA polymerase in the T7 replisome is uncertain, yet it is noteworthy that more than two DNA polymerases are present in several prokaryotic, archaeal, and bacteriophage replisomes, including the bacteriophage T7 system (Geertsema et al, 2014; McInerney et al, 2007; Nossal et al, 2007; Lin et al, 2012). …”

Insight into the cellular involvement of the two reverse gyrases from the hyperthermophilic archaeon Sulfolobus solfataricus

“…Thus, the two reverse gyrases can be distinguished according to their activities at different temperatures. Several studies report that various protein machineries in hyperthermophilic organisms of the Sulfolobus genus are functional at low temperature: the proton pump [ 38 ], the transcription machinery [ 16 ], replication and repair DNA polymerases [ 39 , 40 ] and DNA topoisomerase(s) [ 4 ]. Consequently, we investigated whether both reverse gyrases of S. solfataricus are active at temperatures below 60°C, the lowest temperature previously tested [ 37 ]: we performed topoisomerase assays at temperatures from 45°C to 80°C (Figure 1 ).…”