Biology subjects

Pangeni, S.

Publications and source records attributed to Pangeni, S..

9 recordsLinked to original sources

bioRxiv2026

A superfamily 1 helicase translocates on double-stranded DNA

Replicative and accessory helicases are essential for maintaining genome integrity during DNA replication and repair. Rep helicase belongs to superfamily 1 (SF1), the first helicase family solved at atomic resolution and a longstanding model for how helicases couple ATP hydrolysis to movement on DNA. Using optical tweezers combined with fluorescence imaging, we characterize how Rep translocates and navigates diverse nucleic acid and protein obstacles. We directly observe highly processive translocation on ssDNA (~300 nt/s over >13,000 nt) and show that this directional movement can drive single-stranded DNA-binding proteins (bacterial SSB, archaeal SSB, and eukaryotic RPA) along ssDNA without slowing. On encountering a short duplex barrier ([≤]60 bp), either a DNA duplex or an RNA/DNA hybrid, Rep frequently bypasses it, with termination and unwinding also observed. Unexpectedly, we identify a mode in which Rep transitions from ssDNA onto dsDNA and translocates directionally along the duplex with high processivity(>12,000bp) and at a much higher speed than on ssDNA (~1100 bp/s). AlphaFold3 modeling is consistent with Rep tracking a single strand while keeping the duplex intact. To our knowledge, this is the first demonstration of processive dsDNA translocation by an SF1 helicase, and it broadens models for how translocases operate on DNA during replication and repair.

biophysics↗

bioRxiv2026

WHaloForce enables chemigenetic imaging of molecular tension in living cells and animals

Piconewton forces borne by individual proteins within complex assemblies underlie cell adhesion, migration, and tissue morphogenesis, yet remain difficult to image in living systems. Here, we introduce WHaloForce, a chemigenetic HaloTag-based tension sensor that converts force-dependent relief of tryptophan-mediated dye quenching into a fluorescence lifetime change. Optical tweezers revealed a switch-like unquenching transition near 5 pN, and the sensor responded reversibly to force changes in cells. WHaloForce enabled quantitative tension imaging of diverse force-bearing proteins (vinculin, E-cadherin, -catenin, and laminin) in mammalian cells, mouse tissue, and C. elegans. Bright synthetic dyes made tension measurements possible at endogenous expression levels. In C. elegans, vinculin and laminin showed opposite tension patterns between tissues, revealing distinct force-transmission routes through adhesions and the extracellular matrix. During ovulation, laminin tension accumulated over repeated stretch-relaxation cycles, scaling with cumulative loading history. WHaloForce thus offers a modular platform for imaging spatiotemporal tension patterns in living systems.

cell biology↗

bioRxiv2026

A Modular Framework for Automated Segmentation and Analysis of AFM Imaging of Chromatin Organization

Chromatin organization underlies essential genome functions, but its nanoscale organization remains challenging to capture and quantify with precision. Atomic force microscopy (AFM) offers direct structural readouts of DNA and chromatin, yet translating these rich images into reproducible biological metrics has been limited by the lack of standardized, scalable analysis tools. Here we present DNAsight, an automated analysis framework that integrates machine learning (ML)-based segmentation with modular, base-pair-calibrated quantification of DNA spatial organization, looping, nucleosome spacing, and protein clustering. Applied across diverse chromatin-associated proteins, DNAsight reveals protein-specific organizational signatures, including topology-dependent compaction by integration host factor (IHF), condition-dependent changes in loop-like DNA structures in cohesin-CTCF-precocious dissociation of sisters 5A (PDS5A) reactions, and promoter-driven multimerization of GAGA factor (GAF) clusters. The framework further enables direct extraction of nucleosome spacing distributions from raw AFM images, providing a label-free route to investigate chromatin fiber architecture. Together, these advances establish DNAsight as a generalizable and scalable approach for converting AFM measurements into quantitative insights into the physical principles of chromatin organization. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/708946v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1225883org.highwire.dtl.DTLVardef@1e1041dorg.highwire.dtl.DTLVardef@1d53d18org.highwire.dtl.DTLVardef@9e0a2a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Ultrafast CTCF dynamics control cohesin barrier function

Genomes are organized into chromatin loops through cohesin-mediated extrusion, with CTCF acting as a polar boundary element. As cohesin approaches CTCF at kilobase-per-second speeds, it must rapidly choose whether to stall or bypass. How CTCF encodes this probabilistic decision within a brief encounter window has remained unclear. Here we show that CTCF governs this probabilistic outcome by rapidly sampling a dynamic ensemble of conformations generated by spontaneous rearrangements of its DNA-binding zinc fingers. This ensemble is tuned by DNA sequence, CpG methylation, nearby nucleosomes, and the cohesin regulator PDS5A before cohesin engagement. Upon cohesin binding, PDS5A enhances loop-anchor mechanical stability, reinforcing orientation-dependent boundaries. These findings establish conformational ensemble tuning, rather than static occupancy, as a regulatory principle linking base pair-scale motions to megabase-scale genome organization. One sentence summaryChromatin boundary function is governed not by CTCF occupancy alone, but by a tunable ensemble of DNA-bound conformations that probabilistically gates cohesin capture.

biophysics↗

bioRxiv2025

Mechanistic insights into direct DNA and RNA strand transfer and dynamic protein exchange of SSB and RPA

Single-stranded DNA-binding proteins (SSBs) are essential for genome stability, facilitating replication, repair, and recombination by binding ssDNA, recruiting other proteins, and dynamically relocating in response to cellular demands. Using single-molecule fluorescence resonance energy transfer (smFRET) assays, we elucidated the mechanisms underlying direct strand transfer from one locale to another, protein exchange, and RNA interactions at high resolution. Both bacterial SSB and eukaryotic replication protein A (RPA) exhibited direct strand transfer to competing ssDNA, with rates strongly influenced by ssDNA length. Strand transfer proceeded through multiple failed attempts before a successful transfer, forming a ternary intermediate complex with transient interactions, supporting a direct transfer mechanism. Both proteins efficiently exchanged DNA-bound counterparts with freely diffusing molecules, while hetero-protein exchange revealed that SSB and RPA could replace each other on ssDNA in a length-dependent manner, indicating that protein exchange does not require specific protein-protein interactions. Additionally, both proteins bound RNA and underwent strand transfer to competing RNA, with RPA demonstrating faster RNA transfer kinetics. Competitive binding assays confirmed a strong preference for DNA over RNA. These findings provide critical insights into the dynamic behavior of SSB and RPA in nucleic acid interactions, advancing our understanding of their essential roles in genome stability, regulating RNA metabolism, and orchestrating nucleic acid processes.

molecular biology↗

bioRxiv2024

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

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.

biophysics↗

bioRxiv2024

Rad52 sorts and stacks Rad51 at the DNA junction to promote homologous recombination

Homologous recombination (HR) repairs double-stranded DNA breaks (DSBs). The DSBs are resected to yield single-stranded DNA (ssDNA) that are coated by Replication Protein A (RPA). Rad51 is a recombinase and catalyzes strand invasion and the search for homology. However, it binds to ssDNA with lower affinity than RPA. Thus, mediator proteins such as Rad52/BRCA2 are required to promote Rad51 binding to RPA-coated ssDNA, but the underlying mechanisms remain poorly understood. Saccharomyces cerevisiae Rad52 interacts with Rad51 through two distinct binding modes. We here uncover that the Rad51-binding site in the disordered C-terminus of Rad52 (mode-1) sorts polydisperse Rad51 into discrete monomers. The second Rad51 binding site resides in the ordered N-terminal ring of Rad52 (mode-2), but this interaction occurs at only one position on the ring. In single molecule confocal fluorescence microscopy combined with optical tweezer analysis, we directly visualize filament formation using fluorescent-Rad51. Rad52 catalyzes Rad51 loading onto RPA-coated ssDNA, with a distinct preference for junctions, but no filament growth is observed. Deletion of the C-terminus of Rad52 results in loss of Rad51 sorting and abrogates Rad51 binding to RPA-coated DNA. While BRCA2 and Rad52 are structurally unrelated, many of these functional features are conserved. We describe a concerted Sort & Stack mechanism for mediator proteins in promoting HR.

biochemistry↗

bioRxiv2023

Rapid long-distance migration of RPA on single stranded DNA occurs through intersegmental transfer utilizing multivalent interactions

Replication Protein A (RPA) is a single stranded DNA (ssDNA) binding protein that coordinates diverse DNA metabolic processes including DNA replication, repair, and recombination. RPA is a heterotrimeric protein with six functional oligosaccharide/oligonucleotide (OB) domains and flexible linkers. Flexibility enables RPA to adopt multiple configurations and is thought to modulate its function. Here, using single molecule confocal fluorescence microscopy combined with optical tweezers and coarse-grained molecular dynamics simulations, we investigated the diffusional migration of single RPA molecules on ssDNA under tension. The diffusion coefficient D is the highest (20,000 nucleotides2/s) at 3 pN tension and in 100 mM KCl and markedly decreases when tension or salt concentration increases. We attribute the tension effect to intersegmental transfer which is hindered by DNA stretching and the salt effect to an increase in binding site size and interaction energy of RPA-ssDNA. Our integrative study allowed us to estimate the size and frequency of intersegmental transfer events that occur through transient bridging of distant sites on DNA by multiple binding sites on RPA. Interestingly, deletion of RPA trimeric core still allowed significant ssDNA binding although the reduced contact area made RPA 15-fold more mobile. Finally, we characterized the effect of RPA crowding on RPA migration. These findings reveal how the high affinity RPA-ssDNA interactions are remodeled to yield access, a key step in several DNA metabolic processes. SignificanceReplication Protein A (RPA) binds to the exposed single stranded DNA (ssDNA) during DNA metabolism. RPA dynamics are essential to reposition RPA on ssDNA and recruit downstream proteins at the bound site. Here in this work, we perform a detailed biophysical study on dynamics of yeast RPA on ssDNA. We show that RPA can diffuse on ssDNA and is affected by tension and salt. Our observations are best explained by the intersegmental transfer model where RPA can transiently bridge two distant DNA segments for its migration over long distances. We further dissect the contributions of the trimerization core of RPA and other adjacent RPA molecules on RPA migration. This study provides detailed experimental and computational insights into RPA dynamics on ssDNA.

biophysics↗

bioRxiv2022

Rtt105 configurationally staples RPA and blocks facilitated exchange and interactions with RPA-interacting proteins

Replication Protein A (RPA) binds to single-stranded DNA (ssDNA) and recruits over three dozen RPA-interacting proteins (RIPs) to coordinate multiple aspects of DNA metabolism including DNA replication, repair, and recombination. Rtt105 is a molecular chaperone that regulates nuclear localization of RPA. Whether and how Rtt105 regulates the activities of RPA is poorly understood. Here, we show that Rtt105 binds to multiple DNA binding and protein-interaction domains of RPA and configurationally staples the complex. In the absence of ssDNA, Rtt105 inhibits RPA binding to Rad52, thus preventing spurious binding to RPA-interacting proteins (RIPs). When ssDNA is available, Rtt105 promotes formation of high-density RPA nucleoprotein filaments and dissociates during this process. Free Rtt105 further stabilizes the RPA-ssDNA filaments by inhibiting RPA facilitated exchange. Collectively, our data suggest that Rtt105 sequesters free RPA in the nucleus to prevent untimely RIP interaction, while stabilizing RPA-ssDNA filaments at DNA lesion sites.

biochemistry↗