Publication Date

April 2018

Advisor(s)

Ishita Mukerji

Major

Molecular Biology&Biochemistry

Language

English (United States)

Abstract

The DNA four-way junction (4WJ) is an essential structural intermediate in the process of homologous recombination. Initial experiments have potentially elucidated the intermediates of the 4WJ melting process (Savage, 2017) and have led to the development of a ?pseudo-duplex? model of 4WJ melting. In this thesis, we report the use of multiple spectroscopic techniques to test the validity of this new model and to characterize the global thermodynamic parameters of the melting process. Firstly, the thermodynamic parameters of the 4WJ melting process were characterized using DNA concentration-dependent absorbance spectroscopy. For a more nuanced understanding of the process, fluorescent nucleoside analogs 2-aminopurine (2AP) and 4-amino-6-methyl-8-(2-deoxy-beta-d-ribofuranosyl)-7(8H)-pteridone (6MAP) were incorporated into J3, providing single-base observation of melting behavior. Through our comparison of 2AP and 6MAP reporting in analogous base positions, we concluded that the pseudo-duplex melting behavior previously reported with 6MAP (Savage, 2017) was unique to the junction, and not to the 6MAP probe itself. Additionally, solvent exposure of the analogs was tested through titration with a known fluorescence quencher molecule. These experiments indicated that all probes are similarly exposed to the solvent, suggesting that the fluorescence reported by each probe was not skewed by differing base orientations in duplex. Using data from these experiments, differential scanning calorimetry scans, and coarse-grained simulations, we have outlined a reaction coordinate diagram for the pseudo-duplex model of junction melting. This schematic yields both a qualitative and quantitative characterization of the pseudo-duplex melting model of the DNA four-way junction.

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