Dynamics of HRPA

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Protein dynamics of human RPA-ssDNA and RAD51 presynaptic complexes revealed by single-molecule imaging Replication Protein A is an important single-stranded DNA binding protein involved in many crucial DNA metabolism and repair pathways. Previous research has shown that Saccharomyces cerevisiae RPA can undergo microscopic exchange, allowing it protect ssDNA while rapidly dissociating when downstream factors are present. Here, we report that human RPA can also bind stably and dissociate when required through a similar mechanism. Using total internal reflection fluorescence microscopy, we visualized the dynamics of the human RPA-ssDNA complex when challenged by many different binding proteins, especially how it dissociates during human RAD51 filament assembly. Our technique allows us to observe in real-time how presynaptic complex assembly and disassembly relies on calcium ions, and surprisingly, how the complex (once formed) is unaffected by free RPA. For varying concentrations of hRAD51, we extracted a Hill coefficient of 1.95 ± 0.32. Using the extracted reaction constants, we were able to fit the effects of free RPA on hRAD51 assembly. Our results indicate that although RPA is known to inhibit RAD51 assembly, this only occurs under certain concentration regimes, which is consistent with a competitive inhibitor model where RPA and RAD51 are competing with each other for binding onto the RPA-ssDNA complex. Interestingly, low concentrations of RPA do not stimulate RAD51 assembly on an already RPA-coated ssDNA curtain. These experiments are a basis for further studies of how the competition model between RPA and RAD51 can be modified by recombination mediators such as BRCA2.