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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.

DNA replication is an essential process across all domains of life. Replicative helicases play an integral part in this process by unwinding the duplex DNA to make single-strand template strands available for duplication. While the steric exclusion model of unwinding, where one strand is encircled by the hexameric helicase and the other excluded from the central channel, is widely accepted, the complexities of this process remain unclear. Details of the helicases’ loading and unwinding mechanism(s) are continually being revealed. One such detail is the interaction and role played by the excluded strand.Our group has recently shown that a wrapping interaction between the excluded single-strand of DNA and the outer surface of the helicase is crucial for the unwinding activity of the 3′-5’ MCM helicase from Sulfolobus solfataricus (Sso). Using single-molecule FRET (smFRET), we can now show that this interaction also exists for the hexameric E.coli DnaB (EcDnaB) helicase, which has 5′-3’ polarity, and that similar dynamics are exhibited by both helicases. This suggests that the interaction may be an important component of hexameric helicase unwinding across various helicase superfamilies independent of polarity.We have also investigated the interaction between EcDnaB's inner channel and the encircled-strand using smFRET. A compaction of the encircled strand by the helicase has been suggested based on several crystal structures of hexameric helicases bound to single-strand DNA that exhibits a significant decrease in rise per base. Using EcDnaB, we show that the helicase's binding induces a ‘scrunching’ of the DNA, and that in the absence of ATP, this is a stable interaction. Both excluded-strand wrapping and encircled-strand scrunching are likely critical aspects of replicative helicase unwinding.