Identifier

etd-04072005-110000

Degree

Master of Science (MS)

Department

Biological Sciences

Document Type

Thesis

Abstract

DNA binding by the type I DNA polymerases from Escherichia coli (Klenow) and Thermus aquaticus (Klentaq) has been examined under varying ionic conditions. Klenow and Klentaq both show increased affinity for DNA substrate in the presence of KAcetate over KCl. Ionic linkage relationships were determined for Klenow and Klentaq in KAcetate. Klenow and Klentaq DNA binding show similar ion releases in KAcetate and KCl (~4-5 ions in Klenow, and ~3-4 ions in Klentaq). Interestingly, Klenow and Klentaq exhibit differential ion release in KGlutamate (~4-5 ions in Klentaq and ~2 ions in Klenow). DNA structure dependence of binding was examined for Klentaq. Klentaq showed a preference for double-stranded and primed-template DNA over single-stranded DNA. Klentaq binds double-stranded and primed-template DNA ~300 times tighter than single-stranded DNA at similar salt concentrations. Klentaq has a dramatically reduced ion release when binding single-stranded DNA (~1 ion) versus double-stranded or primed-template DNA (~3-4 ions). To help resolve which DNA binding differences might be related to Klentaq’s thermal stability, an evolutionarily close, but non-thermophilic, homologue of Klentaq (Klendein from Deinococcus radiodurans) has been expressed, purified, and preliminarily examined. Klendein’s circular dichroism (CD) spectrum is similar to Klenow and Klentaq. Further, Klendein’s stability was probed by monitoring the loss of secondary structure by CD upon heating. Klendein has an invariant Tm (32oC) over the pH range 7.5-9.5. A long range goal of the study of the regulation of polymerase-DNA binding by solution conditions will include examining the effects of gravity on the interaction. Although no polymerase experiments have yet been performed in microgravity, as a first step toward this goal, this thesis describes initial equipment construction, modification, and calibration for microgravity measurements. To this end, rates of 2,6-dichloroindophenol (DCIP) reduction by varying ascorbic acid was measured under normal lab conditions and under periods of microgravity, onboard NASA’s KC-135 zero gravity aircraft. This reaction is generally used to calibrate stopped-flow instruments. Reduction of DCIP at pH 6 was determined to be 850-1000 M-1sec-1 under 1g conditions and under microgravity. The reduction of DCIP at pH 9 was determined to be 250-390 M-1sec-1 under 1g conditions and under microgravity.

Date

2005

Document Availability at the Time of Submission

Release the entire work immediately for access worldwide.

Committee Chair

Vince LiCata

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