DNA Structure Dependence of DNA Binding by Escherichia coli and Thermus aquaticus DNA Polymerase I

Abstract

Understanding substrate selection by DNA Polymerase I is important for the balance between repair and replication for this enzyme in vivo. Due to the sequence and structural homologies, all DNA Pol I are considered functional homologues. We have examined the DNA structural preferences of Klenow and Klentaq, the “large fragment” of DNA polymerase I of Escherichia coli and Thermus aquaticus. The binding affinity trend for Klentaq is pt-DNA ≈ ds-DNA >> ss-DNA, while Klenow has long been known to bind ds-DNA very weakly. Temperature dependent examination of Klentaq DNA binding shows that it binds ds-DNA and pt-DNA with parallel affinities across a wide range of temperatures. These non-sequence specific binding proteins exhibit relatively large heat capacity changes (ΔCp) upon DNA binding that reflects significant hydrophobic interactions in the interface of the complex. The magnitude of the ΔCp is also DNA structure dependent as well as being non-homologous for these polymerases. Since these polymerases do not exhibit significant DNA sequence dependence, we conclude that the nature of the “structural site” determines the specific binding thermodynamics. The thermodynamic differences among the different DNA structural substrates for the two polymerases suggest that the in vivo functions of these two supposedly homologous polymerases are different. Funded by the NSF and the Louisiana Biomedical Research Network.