Klenow and Klentaq-DNA Binding: the ‘Glutamate Effect’ is Primarily an Osmotic Effect

Abstract

DNA binding by Klenow (E.coli) and Klentaq (T.aquaticus) DNA polymerases has been studied as a function of monovalent salt concentration, pH and osmotic stress. We previously showed that DNA binding resulted in the net release of 4.5∼5 ions from Klenow and 3∼3.5 ions from Klentaq. Here, we report that Klenow and Klentaq have minimal sensitivity to pH changes, with proton linkages of ∼0.06 and ∼0.3 respectively in KCl. Furthermore, osmotic stress data in KCl indicates 500∼600 waters are released upon binding by both polymerases.Glutamate is the major intracellular anion accumulated in E.coli in the presence of KCl in the external environment. The ‘glutamate effect’ is primarily characterized by an increase in DNA binding affinity when chloride is replaced by glutamate. Some proteins also exhibit decreased ionic linkage in glutamate.Klenow exhibits both aspects of the ‘glutamate effect’. Substituting glutamate for chloride reduces the ionic linkage for Klenow by >50%. The presence of glutamate also increases the proton linkage of Klenow five fold and decreases water release by ∼70% to approximately 150 waters. The dramatic decrease in water release highlights the osmotic nature of the glutamate effect. Glutamate and chloride salts behave as ionic inhibitors of DNA binding but glutamate salts also exhibit an osmotic enhancement effect.While Klentaq's DNA binding affinity is also enhanced by glutamate, its ionic and proton linkages are not altered. The osmotic enhancement is present for Klentaq but it is not as significant in the salt concentration range at which nanomolar Klentaq-DNA binding occurs. E.coli DNA-binding proteins might have evolved to bind tightly at higher salt concentration to utilize the glutamate effect while accumulating intracellular glutamate.