DNA Structure Selectivity of Escherichia coli versus Thermus aquaticus DNA Polymerase I

Abstract

Understanding substrate selection by DNA Polymerase I is important for characterizing the balance between DNA replication and repair for this enzyme in vivo. Due to their sequence and structural similarities, Klenow and Klentaq, the “large fragments” of the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus, are considered functional homologues. We have examined the DNA binding thermodynamics of Klenow and Klentaq to different DNA structures: single‐stranded DNA (ss‐DNA), primed‐template DNA (pt‐DNA), and double‐stranded DNA (ds‐DNA). The DNA binding affinity trend for Klenow from weakest to tightest binding is ds‐DNA < pt‐DNA < ss‐DNA. This is in contrast to Klentaq's DNA binding trend: ss‐DNA < pt‐DNA ≈ ds‐DNA. Both Klenow and Klentaq released more ions when binding to pt‐DNA and ds‐DNA than when binding to ss‐DNA in KCl buffer. ΔCp is the temperature dependence of the enthalpy of a reaction. Both of these non‐sequence specific binding proteins exhibit relatively large heat capacity changes (ΔCp) upon DNA binding. Klenow binding to pt‐DNA and Klentaq binding to pt‐DNA and ds‐DNA show the highest ΔCp values. The thermodynamic differences among the different DNA structural preferences for the two polymerases suggest that the in vivo functions of these two supposedly homologous polymerases are different. This work was supported by NSF and Louisiana Biomedical Research Network.