English (United States)
DNA four-way junctions are branched structures that form between two homologous chromosomes. These junctions play key roles during multiple cellular processes including meiosis and DNA double-stranded break repair. While the structure of these junctions is pivotal to their function in these cellular processes, especially in the uncharacterized mechanism of branch migration, their thermodynamic properties are not well understood. This project investigates the thermodynamic stability of the DNA four-way junction, J3, using the fluorescent nucleoside analogues, 6MAP and 6MI, which provide site-specific information on the junction’s stability. By incorporating these fluorescent probes at different locations in the junction, we are able to investigate the thermodynamic and structural differences of the junction at a base-pair level of specificity. Through this work we have revealed that 6MAP is more sensitive to early melting transitions and 6MI is more sensitive to late melting transitions. The 6MI probe shows that an all-or-nothing model can be used to describe the final steps of the disassociation of the junction, and all four strands have the same stability in the temperature range that encompasses the last steps of this dissociation. The 6MAP probe reveals that in the early melting stages, the X-pseudo-duplex is destabilized at lower temperatures than the H-pseudo-duplex, and that this loss of stability is sequence-dependent. With more information on specific melting patterns and stability, we can begin to make more conclusive models of the mechanism of homologous recombination and double-stranded break repair.
Savage, Rachel Elizabeth, "Investigating the Thermodynamic Stability of DNA Four-Way Junctions Using Fluorescent Nucleoside Analogues" (2017). Honors Theses - All. 1776.
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