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bioRxiv · 10.1101/2024.12.09.627445

The human RAD52 complex undergoes phase separation and facilitates bundling and end-to-end tethering of RAD51 presynaptic filaments

Alshareedah, I.·Pangeni, S.·Dewan, P. A.·Honda, M.·Liao, T.-W.·Spies, M.·Ha, T.
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Abstract

Human RAD52 is a prime target for synthetical lethality approaches to treat cancers with deficiency in homologous recombination. Among multiple cellular roles of RAD52, its functions in homologous recombination repair and protection of stalled replication forks appear to substitute those of the tumor suppressor protein BRCA2. However, the mechanistic details of how RAD52 can substitute BRCA2 functions are only beginning to emerge. RAD52 forms an undecameric ring that is enveloped by eleven [~]200 residue-long disordered regions, making it a highly multivalent and branched protein complex that potentiates supramolecular assembly. Here, we show that RAD52 exhibits homotypic phase separation capacity, and its condensates recruit key players in homologous recombination such as single-stranded (ss)DNA, RPA, and the RAD51 recombinase. Moreover, we show that RAD52 phase separation is regulated by its interaction partners such as ssDNA and RPA. Using fluorescence microscopy, we show that RAD52 can induce the formation of RAD51-ssDNA fibrillar structures. To probe the fine structure of these fibrils, we utilized single-molecule super-resolution imaging via DNA-PAINT and atomic force microscopy and showed that RAD51 fibrils are bundles of individual RAD51 nucleoprotein filaments. We further show that RAD52 induces end-to-end tethering of RAD51 nucleoprotein filaments. Overall, we demonstrate unique macromolecular organizational features of RAD52 that may underlie its various functions in the cell.

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Alshareedah, I., Pangeni, S., Dewan, P. A., Honda, M., Liao, T.-W., Spies, M., Ha, T.. 2024-12-12. The human RAD52 complex undergoes phase separation and facilitates bundling and end-to-end tethering of RAD51 presynaptic filaments. https://doi.org/10.1101/2024.12.09.627445

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