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

Ibraheem Alshareedah; Sushil Pangeni; Paul A Dewan; Masayoshi Honda; Ting-Wei Liao; Maria Spies; Taekjip Ha

doi:10.1101/2024.12.09.627445

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

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

Ibraheem Alshareedah, Sushil Pangeni, Paul A Dewan, Masayoshi Honda, Ting-Wei Liao, Maria Spies and Taekjip Ha

bioRxiv

Cold Spring Harbor Laboratory

12/12/2024

DOI: 10.1101/2024.12.09.627445

PMCID: PMC11661238

PMID: 39713334

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https://doi.org/10.1101/2024.12.09.627445View

Preprint (Author's original)This preprint has not been evaluated by subject experts through peer review. Preprints may undergo extensive changes and/or become peer-reviewed journal articles. Open Access

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.

Details

Title: Subtitle: The human RAD52 complex undergoes phase separation and facilitates bundling and end-to-end tethering of RAD51 presynaptic filaments
Creators: Ibraheem Alshareedah - Boston Children's Museum
Sushil Pangeni - Boston Children's Museum
Paul A Dewan - Boston Children's Museum
Masayoshi Honda - Department of Biochemistry and Molecular Biology, University of Iowa Carver College of Medicine, 51 Newton Road, Iowa City, IA 52242, USA
Ting-Wei Liao - Boston Children's Museum
Maria Spies - Department of Biochemistry and Molecular Biology, University of Iowa Carver College of Medicine, 51 Newton Road, Iowa City, IA 52242, USA
Taekjip Ha - Boston Children's Museum
Resource Type: Preprint
Publication Details: bioRxiv
DOI: 10.1101/2024.12.09.627445
PMID: 39713334
PMCID: PMC11661238
NLM abbreviation: bioRxiv
ISSN: 2692-8205
eISSN: 2692-8205
Publisher: Cold Spring Harbor Laboratory; United States
Language: English
Date posted: 12/12/2024
Academic Unit: Radiation Oncology; Biochemistry and Molecular Biology
Record Identifier: 9984759991802771

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