Document Type

Article

Publication Date

2-21-2003

Abstract

DNA binding properties of the Type 1 DNA polymerases from Thermus aquaticus (Taq, Klentaq) and Escherichia coli (Klenow) have been examined as a function of [KCl] and [MgCl2]. Full-length Taq and its Klentaq "large fragment" behave similarly in all assays. The two different species of polymerases bind DNA with submicromolar affinities in very different salt concentration ranges. Consequently, at similar [KCl] the binding of Klenow is ∼3 kcal/mol (150×) tighter than that of Taq/Klentaq to the same DNA. Linkage analysis reveals a net release of 2-3 ions upon DNA binding of Taq/Klentaq and 4-5 ions upon binding of Klenow. DNA binding of Taq at a higher temperature (60 °C) slightly decreases the ion release. Linkage analysis of binding versus [MgCl2] reports the ultimate release of ∼1 Mg2+ ion upon complex formation. However, the MgCl2 dependence for Klenow, but not Klentaq, shows two distinct phases. In 10 mM EDTA, both polymerase species still bind DNA, but their binding affinity is significantly diminished, Klenow more than Klentaq. In summary, the two polymerase species, when binding to identical DNA, differ substantially in their sensitivity to the salt concentration range, bind with very different affinities when compared under similar conditions, release different numbers of ions upon binding, and differ in their interactions with divalent cations.

Publication Source (Journal or Book title)

Journal of Biological Chemistry

First Page

5694

Last Page

5701

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