All ETDs from UAB

Advisory Committee Chair

David E Graves

Advisory Committee Members

Donald D Muccio

Aaron L Lucius

Herbert C Cheung

Peter E Prevelige

Document Type

Dissertation

Date of Award

2010

Degree Name by School

Doctor of Philosophy (PhD) College of Arts and Sciences

Abstract

G-quadruplex DNAs are higher order nucleic acid structures that have been implicated in the regulation of a number of vital biological processes, including gene transcription and telomeric extension. Guanine rich sequences that are capable of forming G-quadruplex structure are found in the telomeres of chromosomes. The presence of the G-quadruplex structure prevents the binding of telomerase and inhibits the telomeric extension that would result in the immortality of cancer cells. This dissertation describes research that is focused on the biophysical characterization of the G-quadruplex and interactions with a ligand and protein. The anticancer compound, actinomycin D, is demonstrated to bind to both the Na+ and K+ conformations of telomeric G-quadruplex DNA by an end stacking mechanism with favorable binding enthalpies. Complex formation with actinomycin D resulted in changes to both the Na+ and K+ conformations to ligand-bound complexes that had similar structural features and stabilities. End stacking of actinomycin D with G-quadruplex results in a 2:1 G-quadruplex DNA to drug complex that maximizes the stacking interaction of the planar phenoxazone ring on the terminal G-tetrad. In an effort to understand the fundamental properties of G-quadruplex structure and stability, the influence of the adenine bases in the three loops on the energetics and stability of the G-quadruplex were investigated. Adenine and guanine stacking interactions provide important contributions to the overall stability and energetics of the structures. The systematic mutation of adenine to thymine in the three loop sequences results in significant changes in the thermal stability and folding enthalpies. The nucleoprotein, UP1, is demonstrated to recognize and bind with high affinity to G-quadruplex DNA. Upon initial formation of the UP1-G-quadruplex complex the G-quadruplex structure unfolds, allowing the binding of a second UP1 to the exposed TAG site and resulting in a final unfolded G-quadruplex sequence bound with two UP1 molecules. UP1 binding studies with loop mutations revealed that UP1 recognition and initial binding occurs at loop 2. G-quadruplexes with loop 2 mutations revealed a reduction in binding affinity and unfolding by UP1. These results demonstrated that UP1 specifically recognizes G-quadruplex DNA structural motif and the binding energetics are coupled to the unfolding of the G-quadruplex structure.

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