Fusion oncoproteins generated by chromosomal translocations are defining molecular drivers of several pediatric cancers, functioning in part by reprogramming gene expression and disrupting cellular differentiation. Ewing sarcoma, an aggressive cancer of bone and soft tissue, exemplifies this paradigm and is driven in the majority of cases by the oncogenic fusion protein EWS::FLI1, formed by joining the low-complexity domain (LCD) of the RNA-binding protein EWS to the DNA-binding domain (DBD) of the transcription factor FLI1. EWS::FLI1 alters transcriptional and RNA processing programs and assembles into aberrant biomolecular condensates, yet the molecular basis for these fusion-driven assemblies remains poorly understood. Here, we integrate NMR spectroscopy, mutagenesis, biophysical and cellular assays, microscopy, and all-atom molecular dynamics simulations to dissect how sequence features and domain interactions shape the condensate properties of EWS and EWS::FLI1. We find that the EWS LCD undergoes extensive self-association mediated by tyrosine-rich motifs that generate compact intramolecular conformations and robust multivalent networks driving phase separation. The RNA-binding domains of EWS engage the LCD through transient electrostatic and aromatic interactions, modulating condensate dynamics, whereas the FLI1 DBD of the fusion protein directly interacts with the EWS LCD via flexible “wings”. Incorporation of the FLI1 DBD markedly alters the material properties of condensates, producing emergent biophysical behaviors distinct from either parental protein. These altered condensate properties possibly contribute to the cellular dysregulation and toxicity associated with EWS::FLI1, in part through mis-localization and aberrant interactions with native EWS that disrupt normal nuclear organization and RNA processing. Together, these findings define a molecular framework in which weak, multivalent, and chemically specific interactions give rise to the emergent properties of the EWS::FLI1 fusion protein. By linking sequence-encoded features, interdomain crosstalk, and condensate material states, we propose a mechanistic description of how fusion with ETS-family DBDs reprograms the physical behavior of EWS assemblies. These emergent condensate properties provide a potential unifying description of how EWS::FLI1 perturbs transcriptional regulation, RNA metabolism, and nuclear organization in pediatric Ewing sarcoma. David S. Libich, Erich J. Sohn, Kandarp A. Sojitra, Aiola Stoja, Courtney N. Johnson, Jeetain Mittal, Alex A J. Bishop. Emergent condensate behaviors of the EWS::FLI1 fusion protein in Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr B005.

PDS5B (Precocious Dissociation of Sisters 5B) functions in sister chromatid cohesion and genome organization. Interestingly, PDS5B also associates with RAD51, the recombinase required for DNA damage repair by homologous recombination (HR) and the preservation of stressed DNA replication forks against nucleolytic attrition. We show that PDS5B binds dsDNA preferentially over ssDNA and that it enhances RAD51-mediated DNA strand exchange via the capture of dsDNA. PDS5B also acts synergistically with BRCA2-DSS1 to help overcome the interference of RPA in DNA strand exchange and works in conjunction with RAD51 to protect dsDNA against digestion by MRE11-RAD50-NBS1. DNA binding activity resides within the disordered C-terminal region of PDS5B, and testing of a DNA binding mutant provides evidence that this PDS5B attribute underpins protein functions in vitro and in HR and replication fork protection in cells. Our findings thus reveal distinct functions of PDS5B in genome repair and maintenance.

ABSTRACT The breast and ovarian tumor suppressor BRCA1 heterodimerizes with BARD1 to promote DNA double-strand break repair by homologous recombination (HR) and to protect stressed DNA replication forks against nuclease attack. The large, intrinsically disordered central region of BRCA1 harbors binding sites for DNA and multiple repair factors, but its lack of stable structure has hindered mechanistic dissection of these activities. Using biochemical mapping and NMR spectroscopy, we delineate the DNA binding and RAD51 interaction interfaces within this region and construct separation-of-function mutants that selectively ablate each activity. Both DNA binding and RAD51 interaction are required for BRCA1-BARD1 to promote RAD51-mediated DNA strand invasion, and DNA binding also contributes to BLM-DNA2 end resection. These findings provide mechanistic insights into how individual ligand binding activities within BRCA1 contribute to genome maintenance.

Serpine mRNA-binding protein 1 (SERBP1) is an intrinsically disordered RNA-binding protein that regulates translation and ribosome biogenesis through interactions with ribosomes and other molecular complexes. Despite its regulatory importance and implication in cancer development, the molecular basis of SERBP1 RNA recognition remains poorly understood. Here, we characterize the G-quadruplex (G4)-binding properties of SERBP1. Using NMR spectroscopy and biophysical assays, we show that SERBP1 binds parallel G4s, both RNA and DNA, with low micromolar affinity through a conserved mechanism. Molecular dynamics and docking simulations reveal an encircling mechanism in which the RGG box wraps around the G4 while downstream C-terminal serine residues stabilize the complex through hydrogen bonding. Phosphomimetic mutations of key serines disrupt this stabilization and reduce binding affinity, identifying phosphorylation as a regulatory switch for SERBP1 activity. Recognition is driven by G4 topology rather than nucleotide sequence, establishing SERBP1 as a broad-specificity G4-binding protein. We further demonstrate that SERBP1 regulates mTOR expression in glioblastoma cell lines through G4 elements in the mTOR 5' UTR, and that SERBP1 depletion synergizes with mTOR inhibition to reduce cell growth. These results establish SERBP1 as a G4 adaptor protein and represent, to our knowledge, the first detailed characterization of G4 recognition by a fully disordered domain, providing a molecular framework for targeting SERBP1-G4 interactions in cancer.

The BRCA1-associated RING domain protein 1 (BARD1) is the obligate binding partner of the tumor suppressor breast cancer type 1 susceptibility protein (BRCA1) and plays a critical role in maintaining genome integrity. BARD1 contains structured N- and C-terminal domains that mediate heterodimerization with BRCA1, recognition of chromatin marks, and DNA repair functions. Approximately 40

The tumor suppressor protein breast cancer type 1 susceptibility protein (BRCA1) plays a central role in maintaining genome stability through its involvement in DNA damage repair, transcriptional regulation, and cell-cycle control. BRCA1 functions as an obligate heterodimer with its binding partner, the BRCA1-associated RING domain protein 1 (BARD1), to coordinate accurate DNA repair. While the structured N- and C-terminal domains of BRCA1 have been well-characterized, the large central region encoded largely by exon 11 that comprises 80

Homologous recombination (HR) repairs DNA double-strand breaks and stabilizes stressed replication forks, and HR deficiency promotes genome instability and cancer. HR requires assembly of RAD51 nucleoprotein filaments on single-stranded DNA (ssDNA), a process regulated by the human RAD51 paralogs RAD51C, XRCC3, RAD51D and XRCC2. Here, using cryo-electron microscopy, we find that the RAD51-XRCC3-RAD51C complex (RAD51-X3C) assembles into an octamer in which XRCC3 engages the RAD51 DNA-binding surface and RAD51 subunits adopt a misaligned configuration incompatible with filament formation. These features define an autoinhibited RAD51-X3C state that limits nonproductive RAD51 binding to double-stranded DNA or RNA-DNA hybrids while preserving RAD51 availability for ssDNA-dependent strand exchange. We further show that the RAD51D-XRCC2 paralog complex remodels RAD51-X3C into a pentameric RAD51-X3CDX2 assembly by engaging the exposed RAD51C surface and disrupting contacts that stabilize the octamer. This remodeling exposes the RAD51 DNA-binding interface, enhances RAD51-ssDNA filament assembly, and promotes strand exchange on RPA-coated ssDNA, and yields a filament-compatible paralog assembly that integrates into ssDNA-bound RAD51 filaments. Together, these findings establish paralog exchange as a mechanism that converts an autoinhibited RAD51-X3C octamer into an activated RAD51-X3CDX2 pentamer to regulate RAD51 filament formation during HR and replication fork preservation.

Ewing sarcoma, the second most common pediatric bone and soft tissue cancer, is caused by aberrant fusion of the RNA-binding protein EWS (EWS) low-complexity domain (EWSLCD) to the DNA-binding domain of the transcription factor friend leukemia integration 1 (FLI1). The resulting fusion, EWS::FLI1, directly interacts with and engages in a dynamic interplay with EWS that drives tumorigenesis and regulates the function of both proteins. While EWSLCD is known to promote self-association, the role of the RNA-binding domains (RBDs) of EWS, which include arginine-glycine-glycine (RGG) repeat regions and a structured RNA-recognition motif (RRM), remains less well understood. Here, we investigate the interplay between EWSLCD and RBDs using biomolecular condensation assays, microscopy, nuclear magnetic resonance (NMR) spectroscopy, and molecular simulations. Our studies reveal that RBDs differentially influence EWSLCD condensate formation and suggest that electrostatics and polypeptide-chain length likely contribute to this interaction. NMR spectroscopy and molecular dynamics simulations further demonstrate that EWSLCD and the central RNA-binding region, comprising the RRM and RGG2 domains, engage in transient, non-specific interactions that are broadly distributed across both regions and involve diverse residue types. Specifically, tyrosine, polar residues, and proline within EWSLCD preferentially interact with arginine, glycine, and proline residues in the RBD. Atomistic simulations of EWS confirm that the full-length protein exhibits a similar interaction profile with conserved chemical specificity, supporting a model in which a network of weak, distributed interdomain contacts underlies EWS self-association. Together, these findings provide molecular insight into the mechanisms of EWS condensate formation and lay the groundwork for understanding how interdomain interactions regulate EWS and EWS::FLI1 function.

RNA binding protein EWS, a member of the FET (FUS, EWS, TAF15) family, contributes to mRNA biogenesis through roles in transcription, splicing, and RNA transport. Despite evidence linking EWS to spliceosomal complexes, its interactions with spliceosome-associated cyclophilins remain unclear. Here, we describe the first structural and biochemical characterization of the EWS low-complexity domain (EWSLCD) interaction with the spliceosomal cyclophilin PPIL1. Nuclear magnetic resonance (NMR) titration experiments reveal that the proline-rich PxxP motifs of EWSLCD engage the catalytic face of PPIL1, forming low-affinity "fuzzy" complexes. Notably, this interaction is undetected in an EWS construct containing the RNA recognition motif (RRM) and RGG2 domain, suggesting that PxxP accessibility or local context is critical for PPIL1 binding. Phase separation assays demonstrate that PPIL1 is recruited into EWSLCD condensates under physiological salt conditions, while altering condensation properties at lower salt concentrations. These findings support a model where EWS is recruited to spliceosomal cyclophilins, potentially influencing splicing and nascent mRNA processing. This study underscores the functional importance of proline-rich motifs within EWS and highlights the potential of spliceosomal cyclophilins as both catalytic and structural partners. Our work provides a foundation for exploring the mechanism by which cyclophilins modulate EWS biology and for developing novel therapeutic strategies targeting EWS-cyclophilin interactions in cancer.

Homologous recombination (HR) removes DNA double-strand breaks (DSBs) and preserves stressed DNA replication forks. Successful HR execution requires the tumor suppressor BRCA2, which harbors distinct DNA-binding domains (DBDs): one that possesses three oligonucleotide/oligosaccharide-binding (OB) folds (OB-DBD) and another residing in the C-terminal recombinase binding domain (CTRB-DBD). Here, we employ multi-faceted approaches to delineate the contributions of these domains toward HR and replication fork maintenance. We show that OB-DBD and CTRB-DBD confer single-strand DNA (ssDNA)- and dsDNA-binding capabilities, respectively, and that BRCA2 variants mutated in either domain are impaired in their ability to load the recombinase RAD51 onto ssDNA pre-occupied by RPA. While the CTRB-DBD mutant is modestly affected by DNA break repair, it exhibits a strong defect in the protection of stressed replication forks. In contrast, the OB-DBD is indispensable for both BRCA2 functions. Our study thus defines the unique contributions of the two BRCA2 DBDs in genome maintenance.

RNA binding proteins (RBPs) containing intrinsically disordered regions (IDRs) are present in diverse molecular complexes where they function as dynamic regulators. Their characteristics promote liquid-liquid phase separation (LLPS) and the formation of membraneless organelles such as stress granules and nucleoli. IDR-RBPs are particularly relevant in the nervous system and their dysfunction is associated with neurodegenerative diseases and brain tumor development. Serpine1 mRNA-binding protein 1 (SERBP1) is a unique member of this group, being mostly disordered and lacking canonical RNA-binding domains. We defined SERBP1's interactome, uncovered novel roles in splicing, cell division and ribosomal biogenesis, and showed its participation in pathological stress granules and Tau aggregates in Alzheimer's brains. SERBP1 preferentially interacts with other G-quadruplex (G4) binders, implicated in different stages of gene expression, suggesting that G4 binding is a critical component of SERBP1 function in different settings. Similarly, we identified important associations between SERBP1 and PARP1/polyADP-ribosylation (PARylation). SERBP1 interacts with PARP1 and its associated factors and influences PARylation. Moreover, protein complexes in which SERBP1 participates contain mostly PARylated proteins and PAR binders. Based on these results, we propose a feedback regulatory model in which SERBP1 influences PARP1 function and PARylation, while PARylation modulates SERBP1 functions and participation in regulatory complexes.

Serpine mRNA-Binding Protein 1 (SERBP1) is an RNA-binding protein implicated in diverse cellular functions, including translational regulation, tumor progression, and stress response. It interacts with ribosomal subunits, RNA, and proteins involved in stress granules, contributing to processes such as phase separation and epigenetic regulation. Recent studies have shown SERBP1’s role in glioblastoma progression and its involvement in ribosomal regulation. Structurally, SERBP1 contains N- and C-terminal hyaluronan-binding domains, two RG/RGG motifs, and is predicted to be predominantly disordered. Here, we report the backbone resonance assignment and secondary structure propensities of SERBP1’s N-terminal residues (1–149). Using NMR spectroscopy, we identified a stable α-helix (residues 28–40) and transient structural elements. These findings provide insight into the structural features of SERBP1 that may mediate its interactions with ribosomal subunits, RNA, and other binding partners, laying a foundation for future structural studies of its functional mechanisms.

Ewing sarcoma (EwS) is an aggressive cancer of bone and soft tissue that predominantly affects children and young adults. A chromosomal translocation joins the low-complexity domain (LCD) of the RNA-binding protein EWS (EWSLCD) with the DNA-binding domain of Friend leukemia integration 1 (FLI1DBD), creating EWS::FLI1, a potent fusion oncoprotein essential for EwS development and responsible for over 85% of EwS tumors. EWS::FLI1 forms biomolecular condensates in vivo and promotes tumorigenesis through mediation of aberrant transcriptional changes and by interfering with the normal functions of nucleic acid-binding proteins like EWS through a dominant-negative mechanism. In particular, the expression of EWS::FLI1 in EwS directly interferes with the biological functions of EWS leading to alternate splicing events and defects in DNA-damage repair pathways. Though the EWSLCD is capable of phase separation, here we report a direct interaction between FLI1DBD and EWSLCD that enhances condensate formation and alters the physical properties of the condensate. This effect was conserved for three related E-twenty-six transformation-specific (ETS) DNA-binding domains (DBDs) while DNA binding blocked the interaction with EWSLCD and inhibited EWS::FLI1 condensate formation. NMR spectroscopy and mutagenesis studies confirmed that ETS DBDs transiently interact with EWSLCD via the ETS DBDs "wings." Together these results revealed that ETS DBDs, particularly FLI1DBD, enhance EWSLCD condensate formation and rigidity, supporting a model in which electrostatic and structural interactions drive condensate dynamics with implications for EWS::FLI1-mediated transcriptional regulation in EwS.

Antagonistic activities of the 53BP1 axis and the tumor suppressor BRCA1-BARD1 determine whether DNA double-strand breaks (DSBs) are repaired by end joining or homologous recombination. We show that the CTC1-STN1-TEN1 (CST) complex, a central 53BP1 axis component, suppresses DNA end resection by EXO1 and the BLM-DNA2 helicase-nuclease complex but acts by distinct mechanisms in restricting these entities. Whereas BRCA1-BARD1 alleviates the CST-imposed EXO1 blockade, it has little effect on BLM-DNA2 restriction. CST mutants impaired for DNA binding or BLM-EXO1 interaction exhibit a hyper-resection phenotype and render BRCA1-deficient cells resistant to poly(ADP-ribose) polymerase (PARP) inhibitors. Our findings mechanistically define the crucial role of CST in DNA DSB repair pathway choice and have implications for understanding cancer therapy resistance stemming from dysfunction of the 53BP1 axis.

Genomic instability is a hallmark of cancer, enabling the generation of mutations and gross chromosomal rearrangements to drive neoplastic cell transformation and oncogenesis. The BRCA1-BARD1 protein complex acts to eliminate highly toxic DNA double-strand breaks, to ensure the faithful propagation of our genetic blueprint and to suppress cancer development. BRCA1 is a well-described tumor suppressor protein associated with hereditary breast and ovarian cancers as well as sporadic breast cancers, with loss or mutation of BRCA1 leading to triple negative breast cancer and poor patient prognosis. The BRCA1-BARD1 complex promotes homologous recombination (HR), which is the major pathway for the accurate repair of double-strand breaks. However, there is little information regarding the intricate roles fulfilled by BRCA1-BARD1 in this process, or how loss of specific BRCA1-BARD1 functions leads to tumorigenesis. BRCA1 has been previously reported to physically interact with both DNA and RAD51, key factors in HR, but the contributions of the interaction attributes to DNA damage repair remain unknown. Here, we delineate major sites of DNA and RAD51 binding in BRCA1 and use a combination of biochemical and NMR methods to identify the specific residues mediating interactions with these ligands. This has allowed us to develop mutations to ablate BRCA1’s ability to interact with these substrates without affecting BRCA1-BARD1’s interaction with other key DNA repair substrates. Using these BRCA1 mutants impaired for either DNA or RAD51 binding, we have interrogated the contributions of these interaction attributes to BRCA1-BARD1’s function by comparing the activity of WT to mutant BRCA1-BARD1 in biochemical assays to reconstitute various steps of HR. We have found that both DNA and RAD51 binding are indispensable for BRCA1-BARD1’s ability to promote RAD51-mediated D-loop formation, thus helping to delineate the mechanism by which BRCA1 promotes HR. Our studies provide the foundation to determine the functional consequences of cancer mutations in BRCA1-BARD1 and for the development of therapeutic strategies to target HR-deficient tumors. Citation Format: Angela Jasper, Hoang Dinh, Cody M. Rogers, Sameer Salunkhe, Hardeep Kaur, Antoine Baudin, David S. Libich, Patrick Sung. The Mechanistic Role of BRCA1 DNA and RAD51 Binding in DNA Double-Strand Break Repair [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P5-06-06.