Biology subjects

Modesti, M.

Publications and source records attributed to Modesti, M..

10 recordsLinked to original sources

bioRxiv2026

Molecular characterization of MRI/CYREN reveals the Ku binding mode and the role of multimerization in stimulating the activity of NHEJ in DNA repair

Mammalian cells primarily repair DNA double-strand breaks through non-homologous end joining (NHEJ), a pathway that requires the Ku heterodimer, DNA-PKcs, XRCC4 complexed with DNA Ligase 4, and XLF as core components. In addition, several auxiliary proteins are involved in the regulation of NHEJ, whose importance has been underscored recently. Among them, MRI, also known as CYREN, is one of the most important auxiliary proteins. Despite its importance, the structural properties of MRI remain poorly characterized. In this study, we used solution NMR spectroscopy combined with cellular experiments to investigate two isoforms of human MRI (MRI1 and MRI2) at the residue level. Our findings reveal that both isoforms are predominantly disordered, and that the APLF-like Ku-binding motif (A-KBM) of MRI undergoes folding upon binding to the von Willebrand A domain of Ku80 (Ku80vWA). Moreover, an evolutionarily dominant leucine-to-methionine substitution in A-KBM significantly increases binding affinity for Ku80vWA by over 30 times without impacting cellular NHEJ efficiency. Importantly, we identified here a domain that drives MRI multimerization that is required for efficient NHEJ in cellular assays. This work further deciphers the increasingly recognized functional roles of disordered protein regions of the NHEJ machinery.

biophysics↗

bioRxiv2026

Scaling the Dynamics of Coiled Coils

Coiled coils are structural motifs in proteins that play diverse functions. In MRE11-RAD50 (MR) complexes, ATP-driven changes in coiled coils are essential for DNA break sensing. However, coiled coil dynamics and its modulation by protein conformational changes remain unclear, partly due to the lack of quantitative tools. Here, we used high-speed atomic force microscopy (HS-AFM) for real-time visualization of the coiled coil conformational dynamics of individual MR complexes from bacteria and human homologs, and a biomedically relevant variant. The mean square deviation of the end-to-end distance of the coiled coils revealed a power-law scaling with time, conserved across conformational states, homologs, and variants, suggesting a universal dynamic scaling. Coiled coils behave as semi-flexible filaments with strong internal friction, leading to relaxation times that were seconds-long and varied among conformational states and variants. Molecular dynamics simulations indicated that strong friction arose from long-lifetime contacts between coils. Our results suggest that MR complexes modulate the coiled coil dynamics to mediate long-range allosteric and allodynamic communication during DNA repair.

biophysics↗

bioRxiv2026

Structural basis for continuous DNA-end protection during ligation of double-strand breaks in yeast Non-Homologous End-Joining

Non-homologous end joining (NHEJ) repairs DNA double-strand breaks by synapsing and ligating DNA ends. In vertebrates, DNA-PKcs promotes end alignment and processing, yet several eukaryotes - including budding yeast - perform NHEJ without DNA-PKcs, and the underlying mechanism remains unclear. Here we report cryo-electron microscopy structures of reconstituted Saccharomyces cerevisiae NHEJ synaptic complexes assembled on DNA ends bearing either 4-bp terminal microhomologies or blunt termini. On the microhomology substrate with a single 5-P, we capture a ligation-competent short-range complex in which a single Dnl4 catalytic core is fully ordered and engaged at the 5'-adenylated nick. When two 5-P are present, we resolve two predominant, coexisting DNA-aligned protective states in which both Dnl4 DBD-NTD modules occupy the break region with reciprocal geometries, supporting an alternating engagement model for sequential sealing of the two strands while maintaining continuous end association. In contrast, blunt-ended substrates yield a non-aligned protective configuration in which two Dnl4 catalytic modules constrain the DNA ends [~]30 [A] apart in a ligation-incompatible arrangement, providing a structural explanation for slow blunt-end joining in yeast. Together, these structures define architectural and mechanistic principles of DNA-PKcs-independent NHEJ.

molecular biology↗

bioRxiv2025

Near equilibrium unbinding of streptavidin/biotin using single molecule acoustic force spectroscopy.

The dissociation of the streptavidin-biotin (SA-b) bond has been widely characterized using bulk and single molecule force spectroscopy (SMFS) techniques. However, the dissociation rates (koff) from SMFS ([~]10-1 s-1) typically do not align with those from bulk approaches ([~]10-6 - 10-5 s-1), likely because SMFS measurements are conducted far from equilibrium. Near equilibrium SMFS requires high throughput measurements to obtain large enough statistics, and high stability over long time measurements at ultraslow loading force rates, impractical in most SMFS techniques. Here, we developed in-situ force calibration strategies for acoustic force spectroscopy (AFS) to probe the unbinding forces of SA-b in the near equilibrium regime, from 10 pN/s down to 10- 3 pN/s. The resulting koff was in excellent agreement with that from bulk measurements on the very same system. Combined with our previous SA-b data, we covered 15 orders of magnitude in loading rate, setting the ground for SMFS over the widest dynamic range, essential to fully describe the energy landscape of biomolecular processes.

biophysics↗

bioRxiv2025

Ku suppresses RNA-mediated innate immune responses in human cells to accommodate primate-specific Alu expansion.

Ku70 and Ku80 form Ku, a ring-shaped protein that initiates the non-homologous end-joining (NHEJ) DNA repair pathway.1 Specifically, Ku binds to double-stranded DNA (dsDNA) ends and recruits other NHEJ factors (e.g., DNA-PKcs and LIG4). While Ku binds to double-stranded RNA (dsRNA)2 and traps mutated-DNA-PKcs on ribosomal RNA in vivo,3,4 the physiological significance of Ku-dsRNA interactions in otherwise wild-type cells remains elusive. Intriguingly, while dispensable for murine development,5,6 Ku is essential in human cells.7 Despite similar genome sizes, human cells express [~]100-fold more Ku than mouse cells, implying functions beyond NHEJ, possibly through a dose-sensitive interaction with dsRNA, which is [~]100 times weaker than with dsDNA.2,8 While investigating the essentiality of Ku in human cells, we found that depletion of Ku - unlike LIG4 - induces profound interferon (IFN) and NF-kB responses reliant on the dsRNA-sensor MDA5/RIG-I and adaptor MAVS. Prolonged Ku-degradation also activates other dsRNA-sensors, e.g. PKR that suppresses protein translation, and OAS/RNaseL that cleaves rRNAs and eventually induces growth arrest and cell death. MAVS, RIG-I, or MDA5 knockouts suppressed IFN signaling and, together with PKR knockouts, partially rescued Ku-depleted human cells. Ku-irCLIP analyses revealed that Ku binds to diverse dsRNA, predominantly stem-loops in primate-specific Alu elements9 at anti-sense orientation in introns and 3-UTRs. Ku expression rose sharply in higher primates tightly correlating with Alu-expansion (r = 0.94/0.95). Together, our study identified a vital role of Ku in accommodating Alu-expansion in primates by mitigating a dsRNA-induced innate immune response, explaining the rise of Ku levels and its essentiality in human cells.

genetics↗

bioRxiv2024

The KU70-SAP domain has an overlapping function with DNA-PKcs in limiting the lateral movement of KU along DNA

The non-homologous end-joining (NHEJ) pathway is critical for DNA double-strand break repair and is essential for lymphocyte development and maturation. The Ku70/Ku80 heterodimer (KU) binds to DNA ends, initiating NHEJ and recruiting additional factors, including DNA-dependent protein kinase catalytic subunit (DNA-PKcs) that caps the ends and pushes KU inward. The C-terminus of Ku70 in higher eukaryotes includes a flexible linker and a SAP domain, whose physiological role remains poorly understood. To investigate this, we generated a mouse model with knock-in deletion of the SAP domain (Ku70{Delta}SAP/{Delta}SAP). Ku70{Delta}SAP supports KU stability and its recruitment to DNA damage sites in vivo. In contrast to the growth retardation and immunodeficiency seen in Ku70-/- mice, Ku70{Delta}SAP/{Delta}SAP mice show no defects in lymphocyte development and maturation. Structural modeling of KU on long dsDNA, but not dsRNA suggests that the SAP domain can bind to an adjacent major groove, where it can limit KUs rotation and lateral movement along the dsDNA. Accordingly, in the absence of DNA-PKcs that caps the ends, Ku70{Delta}SAP fails to support stable DNA damage-induced KU foci. In DNA-PKcs-/- mice, Ku70{Delta}SAP abrogates the leaky T cell development and reduces both the qualitative and quantitative aspects of residual V(D)J recombination. In the absence of DNA-PKcs, purified Ku70{Delta}SAP has reduced affinity for DNA ends and dissociates more readily at lower concentration and accumulated as multimers at high concentration. These findings revealed a physiological role of the SAP domain in NHEJ by restricting KU rotation and lateral movement on DNA that is largely masked by DNA-PKcs. HighlightKu70 is a conserved non-homologous end-joining (NHEJ) factor. Using genetically engineered mouse models and biochemical analyses, our study uncovered a previously unappreciated role of the C-terminal SAP domain of Ku70 in limiting the lateral movement of KU on DNA ends and ensuring end protection. The presence of DNA-PKcs partially masks this role of the SAP domain.

genetics↗

bioRxiv2023

The RecBC complex protects the nascent DNA in the presence of single-stranded DNA gaps

Following encounter with an unrepaired DNA lesion, replication is halted and can restart downstream of the lesion leading to the formation of a single-stranded DNA (ssDNA) gap. To complete replication, this ssDNA gap is filled in by one of the two lesion tolerance pathways: the error-prone Translesion Synthesis (TLS) or the error-free Homology Directed Gap Repair (HDGR). In the present work, we evidence a new role for the RecBC complex distinct from its canonical function in homologous recombination at DNA double strand breaks. We show that upon lesion encounter RecBC (independently of its catalytic activity and of the RecD subunit) is required to protect the nascent DNA in order to promote efficient lesion bypass. In the absence of RecBC, our data indicate that the nuclease ExoI can access and degrade the nascent DNA, affecting both TLS and HDGR mechanisms. We show that the recruitment of RecBC becomes particularly important at strong blocking lesions, when post-replicatively ssDNA gaps persist and are covered by the single-stranded DNA binding protein (SSB). This protective role of RecBC is reminiscent of the role of BRCA2 in protecting the nascent DNA in human cells, highlighting once again the evolutionary conservation of DNA replication mechanisms across all living organisms. SIGNIFICANCE STATEMENTIn the presence of unrepaired lesions, stalled replication can restart ahead of the lesion creating a single stranded DNA gap. To preserve genome stability, this gap needs to be later filled in by one of the two lesion tolerance pathways, Translesion Synthesis or Homology Directed Gap Repair. In this study, we unveil a role for the RecBC complex in protecting the nascent DNA blocked at the lesion site from degradation by the ExoI nuclease. This protection is essential for an efficient repair of the single-stranded DNA gap. In the absence of RecBC, both lesion tolerance pathways are affected and genome stability is compromised.

molecular biology↗

bioRxiv2023

CRISPR-dependent base editing screens identify separation of function mutants of RADX with altered RAD51 regulatory activity

RAD51 forms nucleoprotein filaments to promote homologous recombination, replication fork reversal, and fork protection. Numerous factors regulate the stability of these filaments and improper regulation leads to genomic instability and ultimately disease including cancer. RADX is a single stranded DNA binding protein that modulates RAD51 filament stability. Here, we utilize a CRISPR-dependent base editing screen to tile mutations across RADX to delineate motifs required for RADX function. We identified separation of function mutants of RADX that bind DNA and RAD51 but have a reduced ability to stimulate its ATP hydrolysis activity. Cells expressing these RADX mutants accumulate RAD51 on chromatin, exhibit replication defects, have reduced growth, accumulate DNA damage, and are hypersensitive to DNA damage and replication stress. These results indicate that RADX must bind RAD51 and promote RAD51 ATP turnover to regulate RAD51 and genome stability during DNA replication.

molecular biology↗

bioRxiv2020

Distinct pathways of homologous recombination controlled by the SWS1-SWSAP1-SPIDR complex

Homology-directed repair (HDR), a critical DNA repair pathway in mammalian cells, is complex, leading to multiple outcomes with different impacts on genomic integrity. However, the factors that control these different outcomes are often not well understood. Here we show that SWS1-SWSAP1-SPIDR controls distinct types of HDR. Despite their requirement for stable assembly of RAD51 recombinase at DNA damage sites, these proteins are not essential for intra-chromosomal HDR, providing insight into why patients and mice with mutations are viable. However, SWS1-SWSAP1-SPIDR is critical for inter-homolog HDR, the first mitotic factor identified specifically for this function. Furthermore, SWS1-SWSAP1-SPIDR drives the high level of sister-chromatid exchange, promotes long-range loss of heterozygosity often involved with cancer initiation, and impels the poor growth of BLM helicase-deficient cells. The relevance of these genetic interactions is evident as SWSAP1 loss prolongs Blm-mutant embryo survival, suggesting a possible druggable target for the treatment of Bloom syndrome.

molecular biology↗

bioRxiv2019

Differential Requirements for the RAD51 Paralogs in Genome Repair and Maintenance in Human Cells

Deficiency in several of the classical human RAD51 paralogs [RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3] is associated with cancer predisposition and Fanconi anemia. To investigate their functions, isogenic disruption mutants for each were generated in non-transformed MCF10A mammary epithelial cells and in transformed U2OS and HEK293 cells. In U2OS and HEK293 cells, viable ablated clones were readily isolated for each RAD51 paralog; in contrast, with the exception of RAD51B, RAD51 paralogs are cell-essential in MCF10A cells. Underlining their importance for genomic stability, mutant cell lines display variable growth defects, impaired sister chromatid recombination, reduced levels of stable RAD51 nuclear foci, and hypersensitivity to mitomycin C and olaparib. Altogether these observations underscore the contributions of RAD51 paralogs in diverse DNA repair processes, and demonstrate essential differences in different cell types. Finally, this study will provide useful reagents to analyze patient-derived mutations and to investigate mechanisms of chemotherapeutic resistance deployed by cancers.

cell biology↗