Biology subjects

Libich, D. S.

Publications and source records attributed to Libich, D. S..

9 recordsLinked to original sources

bioRxiv2026

BRCA1 promotes homologous recombination through separable DNA and RAD51 binding activities within its disordered region.

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.

biochemistry↗

bioRxiv2026

Intrinsically disordered SERBP1 regulates translation through topology-driven G-quadruplex recognition.

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. Significance StatementSERBP1 is overexpressed in multiple cancers and regulates key cellular processes, yet how it recognizes its RNA targets has remained unclear. We show that SERBP1 binds G-quadruplex structures through an encircling mechanism in which its RGG box and adjacent C-terminal serine residues lock around the G4 topology, and that phosphorylation of these serines acts as a switch to modulate binding. Recognition is driven by G4 topology rather than nucleotide sequence, positioning SERBP1 as a general G4 adaptor that recruits helicases to resolve these structures and license translation. We demonstrate this principle in glioblastoma, where SERBP1 regulates mTOR expression through direct recognition of G4 elements in the mTOR 5 UTR, and its depletion synergizes with mTOR inhibition. These findings provide the first molecular characterization of G4 recognition by a fully disordered domain and establish a direct link between SERBP1-G4 interactions and cancer-relevant translational regulation.

biophysics↗

bioRxiv2025

RAD52 and RPA act in a concert promoting inverse RNA strand exchange

Recent studies in eukaryotes have revealed an important role of RNA in DNA repair and identified the RAD52 protein as a central player in RNA-dependent repair of DNA. In vitro, RAD52 promotes inverse RNA strand exchange between dsDNA and homologous RNA. This reaction is strongly stimulated by the RAD52 partner, replication protein A (RPA). Here, using NMR and biochemical methods we investigated the mechanism of this stimulation. We identified two RPA-binding sites in the unstructured RAD52 C-terminal domain (CTD), which mediate interaction with RPA70 and RPA32 subunits. These interactions are critical for stimulation of inverse RNA strand exchange. Furthermore, we showed that stimulation of inverse RNA strand exchange requires formation of an RPA-RNA complex that strengthens the RPA-RAD52 interaction and serves to deliver RNA to the RAD52-dsDNA complex for strand exchange. These results elucidate the mechanism of novel inverse RNA strand exchange activity of RAD52 and the role of RAD52-RPA interaction in RNA-dependent DNA repair.

biochemistry↗

bioRxiv2025

Molecular Basis of EWS Interdomain Self-Association and Its Role in Condensate Formation

Ewing sarcoma, the second most common pediatric bone and soft tissue cancer, is caused by aberrant fusion of the EWS low-complexity domain (EWSLCD) to the DNA-binding domain of the transcription factor 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 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, 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. SIGNIFICANCEThis study shows that the RNA-binding regions of the RNA-binding protein EWS subtly tune biomolecular condensation through numerous transient contacts with the low complexity domain that is common to both EWS and the oncogenic fusion EWS::FLI1. These results provide insight into condensate formation of EWS, which may be useful for understanding the oncogenic mechanisms of EWS::FLI1 and broadly, into pathogenic biomolecular condensate formation.

biophysics↗

bioRxiv2024

Distinct roles of the two BRCA2 DNA binding domains in DNA damage repair and replication fork preservation

Homologous recombination (HR) is a highly conserved tool for the removal of DNA double-strand breaks (DSBs) and the preservation of stalled and damaged DNA replication forks. Successful completion of HR requires the tumor suppressor BRCA2. Germline mutations in BRCA2 lead to familial breast, ovarian, and other cancers, underscoring the importance of this protein for maintaining genome stability. BRCA2 harbors two distinct DNA binding domains, one that possesses three oligonucleotide/oligosaccharide binding (OB) folds (known as the OB-DBD), and with the other residing in the C-terminal recombinase binding domain (termed the CTRB-DBD) encoded by the last gene exon. Here, we employ a combination of genetic, biochemical, and cellular approaches to delineate contributions of these two DNA binding domains toward HR and the maintenance of stressed DNA replication forks. We show that OB-DBD and CTRB-DBD confer ssDNA and dsDNA binding capabilities to BRCA2, respectively, and that BRCA2 variants mutated in either DNA binding domain are impaired in the ability to load the recombinase RAD51 onto ssDNA pre-occupied by RPA. While the CTRB-DBD mutant is modestly affected for HR, 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 DNA binding domains in genome maintenance.

biochemistry↗

bioRxiv2024

SERBP1 interacts with PARP1 and is present in PARylation-dependent protein complexes regulating splicing, cell division, and ribosome biogenesis

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 SERBP1s interactome, uncovered novel roles in splicing, cell division and ribosomal biogenesis, and showed its participation in pathological stress granules and Tau aggregates in Alzheimers 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.

genomics↗

bioRxiv2023

Insights into Molecular Diversity within the FET Family: Unraveling Phase Separation of the N-Terminal Low Complexity Domain from RNA-Binding Protein EWS

The FET family proteins, which includes FUS, EWS, and TAF15, are RNA chaperones instrumental in processes such as mRNA maturation, transcriptional regulation, and the DNA damage response. These proteins have clinical significance: chromosomal rearrangements in FET proteins are implicated in Ewing family tumors and related sarcomas. Furthermore, point mutations in FUS and TAF15 are associated with neurodegenerative conditions like amyotrophic lateral sclerosis and frontotemporal lobar dementia. The fusion protein EWS::FLI1, the causative mutation of Ewing sarcoma, arises from a genomic translocation that fuses the low-complexity domain (LCD) of EWS (EWSLCD) with the DNA binding domain of the ETS transcription factor FLI1. This fusion not only alters transcriptional programs but also hinders native EWS functions like splicing. However, the precise function of the intrinsically disordered EWSLCD is still a topic of active investigation. Due to its flexible nature, EWSLCD can form transient interactions with itself and other biomolecules, leading to the formation of biomolecular condensates through phase separation - a mechanism thought to be central to the oncogenicity of EWS::FLI1. In our study, we used paramagnetic relaxation enhancement NMR, analytical ultracentrifugation, light microscopy, and all-atom molecular dynamics (MD) simulations to better understand the self-association and phase separation tendencies of EWSLCD. Our aim was to elucidate the molecular events that underpin EWSLCD-mediated biomolecular condensation. Our NMR data suggest tyrosine residues primarily drive the interactions vital for EWSLCD phase separation. Moreover, a higher density and proximity of tyrosine residues amplify the likelihood of condensate formation. Atomistic MD simulations and hydrodynamic experiments revealed that the tyrosine-rich N and C-termini tend to populate compact conformations, establishing unique contact networks, that are connected by a predominantly extended, tyrosine-depleted, linker region. MD simulations provide critical input on the relationship between contacts formed within a single molecule (intramolecular) and inside the condensed phase (intermolecular), and changes in protein conformations upon condensation. These results offer deeper insights into the condensate-forming abilities of the FET proteins and highlights unique structural and functional nuances between EWS and its counterparts, FUS and TAF15.

biophysics↗

bioRxiv2022

The oncogenic fusion protein EWS-FLI1 promotes premature ageing of biomolecular condensates by catalyzing fibril formation.

Ewing sarcoma (EwS) is an aggressive pediatric cancer of bone and soft tissue. A chromosomal translocation that joins the low-complexity domain of EWS (EWSLCD) with the DNA-binding domain of FLI1 (FLI1DBD) creates EWS-FLI1, a fusion oncoprotein essential for EwS development and accounts for 85% of all EwS cases. EWS-FLI1 acts as an aberrant transcription factor and interferes with the normal functions of nucleic acid-binding proteins via multivalent interactions and biomolecular condensation. The FLI1DBD was found to directly interact with the EWSLCD causing enhanced phase separation and induced hardening of EWSLCD condensates. Three related ETS DBDs (ERG, ETV1 and PU.1) also induced EWSLCD condensate hardening. DNA binding blocked the interaction with the EWSLCD, and NMR spectroscopy confirmed that ETS DBDs interact with EWSLCD via the DNA-binding interface. Our results provide a physical basis for the dominant-negative effect EWS-FLI1 exerts on EWS and highlight the need for further investigations of the FLI1DBD-EWSLCD interaction in vivo.

biophysics↗

bioRxiv2021

Defining function of wild-type and patient specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

In embryonal rhabdomyosarcoma (ERMS) and generally in sarcomas, the role of wild-type and loss or gain of function TP53 mutations remains largely undefined. Eliminating mutant or restoring wild-type p53 is challenging; nevertheless, understanding TP53 effects on tumorigenesis remains central to realizing better treatment outcomes. In ERMS, >70% of patients retain wild-type TP53, yet TP53 mutations when present in tumors are associated with poor prognosis. Employing a kRASG12D-driven ERMS tumor model and newly generated tp53 null (tp53-/-) zebrafish, we define both wild-type and patient-specific TP53 mutant effects on tumorigenesis. We demonstrate that tp53 is a major suppressor of tumor initiation, where tp53 loss expands tumors initiation from <35% to >97% of animals. Next, characterizing three patient-specific mutants finds that TP53C176F partially retains wild-type p53 apoptotic activity that can be exploited, while the TP53P153{Delta} and TP53Y220C mutants define two structural mutations that predispose to head musculature ERMS.

cancer biology↗