Author ORCID Identifier

Defense Date


Document Type


Degree Name

Doctor of Philosophy



First Advisor

Dr. Soma Dhakal


DNA breaks are inevitable as they mainly occur due to cells’ own reactive oxygen species (ROS). While DNA breaks can be single-stranded or double-stranded, the double-stranded DNA (dsDNA) breaks are more dangerous. If such damage is not repaired, it can lead to genetic instability and serious health issues including cancers. One way dsDNA breaks can be repaired is via a process called homologous recombination (HR), which involves several DNA-binding proteins. Therefore, to have a better insight into the repair mechanism and origin of repair defects, we need a better understanding of how these proteins interact with DNA itself and DNA intermediates of the HR process such as Holliday junctions (HJs). The HJ is a four-way branched structure formed between two homologous DNA molecules during exchange of nucleotide sequences, which is a central intermediate of the DNA repair via the HR process. The HJs are eventually resolved into regular dsDNA molecules by a set of proteins called HJ resolvases. Therefore, knowledge of the binding interaction of these proteins and HJ can provide critical insights into the origin of diseases and potential treatments. Although the HR process has been the subject of intensive study for more than three decades, the complex and dynamic nature of protein–protein and protein–DNA interactions during HR present a significant challenge for determining the molecular mechanism(s) of the process. This knowledge gap is largely because of the dynamic interactions between HR proteins and DNA, which is difficult to capture by routine biochemical or structural biology methods. One remedy for this problem is the employment of single molecule techniques such as single-molecule fluorescence microscopy and optical tweezers. These tools provide unique ways of probing these complex and dynamic interactions at high spatiotemporal resolution, revealing mechanistic insights of the process. However, for single molecule fluorescence microscopy experiments we needed a single molecule total internal reflection fluorescence microscope which we custom built. Using single-molecule fluorescence resonance energy transfer (smFRET) and ensemble analyses, we recently investigated the binding interaction between the HJ and RuvA – a prokaryotic protein that recognizes the HJ and initiates its resolution by forming a resolvase protein complex called RuvABC. Using the HJ labeled with a donor and acceptor fluorophores to enable smFRET, we show that RuvA stably binds to a specific conformation of the HJ, halting its conformational dynamics. Further, the FRET experiments in different ionic environments created by Mg2+ ions suggest that RuvA binds to the HJ via electrostatic interaction. These insights led us to a follow up study looking at the mechanical stability of the RuvA-HJ complex. We have recently developed an optical tweezers-based single-molecule manipulation assay to detect the formation of protein-HJ complexes, which we implemented to study the RuvA-HJ complex and determined its mechanical and thermodynamic properties in a manner that would be impossible with traditional ensemble techniques. We found that the binding of RuvA increases the unfolding force (Funfold) of the HJ by ~2-fold, demonstrating that the RuvA protein stabilizes the junction. Further, the analysis of F-X curves. To our surprise, we also observed that RuvA provides stabilization that permits refolding of the HJ at a force higher than the unfolding force of the HJ without protein. This observation suggests that RuvA stays bound to the DNA construct even after unfolding of the HJ motif, may serve as a nucleation site for HJ refolding, and reduces the energy required for HJ refolding. Together, using high-resolution single-molecule studies we have revealed several molecular insights of the binding interaction between aforementioned proteins and HJ furthering our understating of their roles in the critical HR process. The better the HR process is understood the more likely the scientific community will be able to develop ways of modulating this process for the treatment of recombination related diseases.


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