Identifier

etd-11112009-111519

Degree

Doctor of Philosophy (PhD)

Department

Biological Sciences

Document Type

Dissertation

Abstract

In this study, it is shown that the large fragments of the type I DNA polymerase from E.coli (Klenow) and T.aquaticus (Klentaq) display enhanced DNA binding affinity in glutamate vs. chloride. Across the relatively narrow salt concentration ranges often used to obtain salt linkage data, Klenow also displays an apparently decreased linked ion released (¦¤nions) in Kglutamate vs. KCl while Klentaq does not display such an effect. The osmotic stress technique reveals that Klenow and Klentaq DNA binding is associated with the release of ~500 to 600 waters in KCl. For both proteins, replacing chloride with glutamate results in a 70% reduction in the hydration change upon DNA binding (to ~150-200), highlighting glutamate's osmotic role. To further examine this osmotic effect of glutamate, the salt-DNA binding linkages were extended up to 2.5 M Kglutamate. Consequently, a reversal of the salt linkage is observed above 800mM for both proteins. Salt addition titrations confirmed that rebinding of salt displaced polymerase to DNA occurs beyond 1M [Kglutamate]. Non linear analysis of the biphasic salt linkage indicates that the osmotic role of glutamate is responsible for the reversed linkage and allows the quantitative dissection of the ionic and osmotic behaviors. The similar effect of glutamate on the two polymerases results in a relatively constant affinity difference (¦¤¦¤Gobind(KLN-KTQ)¡Ö-3kcal/mol) throughout the entire salt range. The catalytic activity of both polymerases persists into higher [Kglutamate] than [KCl]. However, the re-association of the proteins on the DNA in high Kglutamate does not result in enhanced catalytic activity. These data represent only the second documentation of an apparent reversed salt linkage for a protein-DNA interaction. This unusual behavior is quantitatively accounted for by a shifting balance of ionic and osmotic effects of the glutamate anion.

Date

2009

Document Availability at the Time of Submission

Student has submitted appropriate documentation to restrict access to LSU for 365 days after which the document will be released for worldwide access.

Committee Chair

LiCata, Vince J.,

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