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Joseph M. Knee; Ishita Mukerji






The ability to observe structure and dynamics of single residues in an oligomer is critical when investigating the function of DNA-binding proteins. The development of fluorescent nucleoside analogs, has greatly improved the amount of information available from both steady-state and time-resolved fluorescence experiments. The significant reduction in quantum yield observed when probes are incorporated into either an oligomer or a duplex limits their potential application and use. The focus of this thesis is on the phototphysical characterization and application of the guanine analogue 6-methylisoxanthopterin (6-MI), specifically in the context of sequence specific fluorescence enhancement. While investigating the photophysical properties of this nucleoside analog; we discovered a pentamer DNA sequence (ATFAA), which exhibits enhanced fluorescence (in either orientation) upon formation of duplex DNA. After extensive characterization of this unique sequence we hypothesized the enhanced fluorescence was the result of a sterically hindered conformation of 6-MI. In this sequence 6-MI has reduced mobility preventing access to the normal non-radiative pathways that an analog experiences when incorporated into a polymer of nucleotides. We exploited the high quantum yield and well characterized pentamer system to examine the interaction between nucleoid associated protein HU and the structural specific overhang construct.

HU is a non-sequence specific DNA/RNA binding protein, which exhibits a strong preference for various distortions in DNA; nicks, gaps, cruciform, and overhangs. The key structural and dynamic elements important in the mechanism of HU structural specificity remain unknown. An important advance is to identify the solution state geometry of the HU-overhang complex and understand the role of nucleic acid topology in HU specificity. We developed a model of the HU-overhang complex based on using the fluorescent base analog 6-methylisoxanthopterin (6-MI) to examine local base dynamics and Förster resonance energy transfer to investigate the global DNA structure by generating distance constraints for the HU-overhang complex in solution. HU binding induces a ~120° bend to the overhang construct. We hypothesize the high affinity complex, KD is ~300 pM, forms when the HU α subunit recognizes the unique conformational dynamics of bases at the junction, while ~9bp away the HU β subunit interacts with single-stranded DNA.



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