Complex Temperature Dependent Equilibria Dictate DNA Polymerase Exchange Processes during Synthesis

Abstract

Most organisms encode for multiple DNA polymerases with similar substrate affinities, but vastly different fidelities. Proper genomic maintenance by the high fidelity (PolB1) and lesion bypass polymerases (PolY) from Sulfolobus solfataricus involves a complex solution equilibria of protein complexes and specific recognition of appropriate DNA substrates. Using isothermal titration calorimetry (ITC) and temperature dependent electrophoretic mobility shift assays (tEMSAs), we have found differences in oligomeric assemblies of Dpo1 and Dpo4 on DNA that include unusually strong temperature dependence changes in heat capacity (ΔCp), which switch from positive to negative values as temperature increases over a 60°C range. The thermodynamic data suggests that binding of PolB1 to DNA is favored over PolY by changes in solution multiequilibria (monomer-oligomer) with temperature that influence ΔCp values. We have also followed polymerase exchange events between PolB1 and PolY and themselves and with the sliding clamp, PCNA, during active DNA synthesis using ensemble kinetic and fluorescence resonance energy transfer (FRET) assays. Surprisingly, the assembled PolB1 holoenzyme complex synthesizes DNA distributively and with low processivity, unlike most other well-characterized DNA polymerase holoenzyme complexes. Exchange between polymerases is temperature and concentration dependent process that is orchestrated by several contacts with PCNA. Simultaneous binding of PolB1 and PolY1 to PCNA allows for dynamic exchange of polymerase active sites during replication and lesion bypass synthesis. Taken together, our results distinguish between specific thermodynamic parameters including temperature dependent coupled equilibria and structural complementary; kinetic processes; and protein contacts that direct binding for uninterrupted but dynamic DNA replication and repair processes at high temperatures.