Title
The glutamate effect on DNA binding by pol I DNA polymerases: osmotic stress and the effective reversal of salt linkage
Document Type
Article
Publication Date
8-13-2010
Abstract
The significant enhancing effect of glutamate on DNA binding by Escherichia coli nucleic acid binding proteins has been extensively documented. Glutamate has also often been observed to reduce the apparent linked ion release (Deltan(ions)) upon DNA binding. In this study, it is shown that the Klenow and Klentaq large fragments of the Type I DNA polymerases from E. coli and Thermus aquaticus both display enhanced DNA binding affinity in the presence of glutamate versus chloride. Across the relatively narrow salt concentration ranges often used to obtain salt linkage data, Klenow displays an apparently decreased Deltan(ions) in the presence of Kglutamate, while Klentaq appears not to display an anion-specific effect on Deltan(ions). Osmotic stress experiments reveal that DNA binding by Klenow and Klentaq is associated with the release of approximately 500 to 600 waters in the presence of KCl. For both proteins, replacing chloride with glutamate results in a 70% reduction in the osmotic-stress-measured hydration change associated with DNA binding (to approximately 150-200 waters released), suggesting that glutamate plays a significant osmotic role. Measurements of the salt-DNA binding linkages were extended up to 2.5 M Kglutamate to further examine this osmotic effect of glutamate, and it is observed that a reversal of the salt linkage occurs above 800 mM for both Klenow and Klentaq. Salt-addition titrations confirm that an increase of [Kglutamate] beyond 1 M results in rebinding of salt-displaced polymerase to DNA. These data represent a rare documentation of a reversed ion linkage for a protein-DNA interaction (i.e., enhanced binding as salt concentration increases). Nonlinear linkage analysis indicates that this unusual behavior can be quantitatively accounted for by a shifting balance of ionic and osmotic effects as [Kglutamate] is increased. These results are predicted to be general for protein-DNA interactions in glutamate salts.
Publication Source (Journal or Book title)
Journal of molecular biology
First Page
223
Last Page
38
Recommended Citation
Deredge, D. J., Baker, J. T., Datta, K., & Licata, V. J. (2010). The glutamate effect on DNA binding by pol I DNA polymerases: osmotic stress and the effective reversal of salt linkage. Journal of molecular biology, 401 (2), 223-38. https://doi.org/10.1016/j.jmb.2010.06.009