Theses Doctoral

Single-molecule observations of hRPA, RAD51, and RAD52 on single-stranded DNA

Ma, Chu Jian

Deoxyribonucleic acid (DNA), like the hard drive in a computer, stores all the essential information for cell function and survival in nearly every single cell in our body. Four different bases are the building blocks of DNA that encode all the messages. As each cell divides, it must pass down its entire genomic DNA to both of its daughter cells. Given the vast amount of data that exists, many errors occur naturally every day and threaten the integrity of this biological hard drive. Normal cells are equipped with many repair tools to quickly and effectively respond to the lesions. When some of these errors disrupt the tightly regulated cell division, cells could undergo changes like an increase in the rate of division that eventually lead to cancer. One type of DNA damage that has a high propensity to cause genetic instability is the double-stranded break (DSB). Therefore, mechanisms that repair DSB are an important area of study in the fight against cancer and cancer causing syndromes. One of these repair pathways is homologous recombination (HR), which uses homologous sequences from either a sister chromatid or a homologue to fill in the information lost during a DSB. This homology pairing reaction requires a class of ATP-dependent proteins known as recombinases, with RAD51 being the one for humans. During HR, the early stages before pairing involve resection of the newly generated DSB ends to generate single-stranded DNA (ssDNA) overhangs, which are protected from degradation by replication protein A (RPA). RAD51 needs to displace the RPA from ssDNA and form a filament (the presynaptic complex) in order to initiate homology search. This process can be sped up by recombination mediators, which act to help RAD51 overcome the strong affinity of RPA for ssDNA that inhibits RAD51 binding and filament formation. Although Rad52 is the most important mediator in budding yeast, human RAD52 does not appear to have mediator function despite a high level of structural conservation. However, human RAD52 mediates ssDNA annealing and its deficiency is synthetic lethal with several important recombination proteins. Here, I use the single-molecule imaging technique of DNA curtains to visualize in real-time the competition and cooperativity between RPA, RAD52, and RAD51 on ssDNA through fluorescent labeling of RPA and RAD52. Using ssDNA curtains, I examine the conservation of facilitated dissociation from budding yeast to humans and show it does not require species-specific contacts. I also monitor the interactions of RAD52 with the RPA-ssDNA and find another point of conservation in the ability of RAD52 to upregulate the stability of RPA on ssDNA concerning facilitated dissociation. These RAD52-RPA-ssDNA complexes are long-lived; however, they are effectively displaced by RAD51 during filament assembly and do not re-bind appreciably to the RAD51 filament. Although RAD51 can still assemble on RAD52-RPA-ssDNA, I observe a significant inhibition on its nucleation (the first step in filament formation), but not elongation, by the presence of free RPA in solution. As DNA curtains allow efficient exchange of buffers in the micro-fluidic chambers while keeping ssDNA molecules tethered, I am able to follow individual DNA molecules overtime as they undergo different binding and filament assembly and disassembly reactions.


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More About This Work

Academic Units
Cellular, Molecular and Biomedical Studies
Thesis Advisors
Greene, Eric C.
Ph.D., Columbia University
Published Here
October 10, 2017