Biology subjects

Rudnizky, S.

Publications and source records attributed to Rudnizky, S..

10 recordsLinked to original sources

bioRxiv2026

A Distributed Architectural Code Governs DNA Repair

Repair of DNA double-strand breaks (DSBs) depends on large-scale changes in nuclear shape and genome organization. How damage signaling coordinates such remodeling remains unclear. We find that the human DNA damage response (DDR) couples interdependent, temporally ordered tubulin modifications to nuclear membrane and lipid regulation, driving nuclear reshaping (nucleomorphosis) through DSB-capturing nuclear envelope tubules (dsbNETs). Tubulin detyrosination by vasohibins 1/2 (VASH1/2) and acetylation by -tubulin acetyltransferase 1 (ATAT1) promote dsbNET formation and support both homologous recombination and non-homologous end joining. Microtubule-dependent processes also promote the phosphorylation-dependent inactivation of the membrane stress-sensing lipid regulator CTP cytidylyltransferase- (CCT), which typically restrains baseline nuclear lipid droplets (nLDs) and dsbNETs while supporting ATR signaling. Damage-induced nLD formation requires diacylglycerol acyltransferases 1/2 (DGAT1/2) and supports dsbNETs. Our findings reveal a distributed architectural code spanning interconnected cytoskeletal, membrane, and lipid regulators. This code governs nucleomorphosis and its role in DNA repair and genome stability.

molecular biology↗

bioRxiv2026

Tunable kinetic destabilization governs RNA polymerase passage through a DNA-bound transcription factor

Eukaryotic transcription factors recognize short motifs, creating abundant binding sites within gene bodies and potential collisions with elongating RNA polymerases, yet how such encounters are resolved remains unclear. Here, we use optical tweezers to monitor RNA polymerase transcription through DNA-bound Egr-1, a zinc-finger transcription factor. Using DNA-fluctuation suppression as a readout of polymerase arrival, we show that Egr-1 delays elongation in an orientation- and rNTP-dependent manner, whereas force measurements indicate that RNAP does not bypass the TF by mechanical eviction. Instead, RNAP destabilizes the Egr-1-DNA complex over a short, structured interaction zone, increasing TF dissociation non-monotonically with distance. Monte Carlo simulations incorporating these kinetic changes recapitulate passage-time distributions. CpG methylation shortens Egr-1 residence time and largely eliminates the TF-dependent delay, suggesting a role for gene-body methylation in reducing kinetic barriers to elongation. These results reveal DNA-bound TFs as tunable barriers that locally shape transcription elongation.

biophysics↗

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↗

bioRxiv2021

Single molecule characterization of the binding kinetics of a transcription factor and its modulation by DNA sequence and methylation

The interaction of transcription factors with their response elements in DNA is emerging as a highly complex process, whose characterization requires measuring the full distribution of binding and dissociation times in a well-controlled assay. Here, we present a single-molecule assay that exploits the thermal fluctuations of a DNA hairpin, to detect the association and dissociation of individual, unlabeled transcription factors. We demonstrate this new approach by following the binding of Egr1 to its consensus motif and the three binding sites found in the promoter of the Lhb gene, and find that both association and dissociation are modulated by the 9 bp core motif and the sequences around it. In addition, CpG methylation modulates the dissociation kinetics in a sequence and position-dependent manner, which can both stabilize or destabilize the complex. Together, our findings show how variations in sequence and methylation patterns synergistically extend the spectrum of a proteins binding properties, and demonstrate how the proposed approach can provide new insights on the function of transcription factors.

biophysics↗

bioRxiv2020

Extended and dynamic linker histone-DNA interactions control chromatosome compaction

Chromatosomes play a fundamental role in chromatin regulation, but a detailed understanding of their structure is lacking, partially due to their complex dynamics. Using single-molecule DNA unzipping with optical tweezers, we reveal that linker histone interactions with DNA are remarkably extended, with the C-terminal domain binding both DNA linkers as far as ~ {+/-}140 bp from the dyad. In addition to a symmetrical compaction of the nucleosome core governed by globular domain contacts at the dyad, the C-terminal domain compacts the nucleosomes entry and exit. These interactions are dynamic, exhibiting rapid binding and dissociation, sensitive to phosphorylation of a specific residue, and crucial to determining the symmetry of the chromatosomes core. Extensive unzipping of the linker DNA, which mimics its invasion by motor proteins, shifts H1 into an asymmetric, off-dyad configuration and triggers nucleosome decompaction, highlighting the plasticity of the chromatosome structure and its potential regulatory role.

biophysics↗

bioRxiv2017

Auxiliary ATP binding sites power rapid unwinding by RecBCD

RecBCD, responsible for the initiation of double stranded break repair in bacteria, is a processive DNA helicase with an unwinding rate approaching [~]1,600 bp{middle dot}s-1. The mechanism enabling RecBCD to achieve such fast unwinding rate is not known. We employed a combination of equilibrium and time-resolved binding experiments, and ensemble and single molecule activity assays to uncover the molecular mechanism underlying RecBCDs rapid catalysis. We report the existence of auxiliary binding sites, where ATP binds with lower affinity and with distinct chemical interactions as compared to the known catalytic sites. The catalytic rate of RecBCD is reduced both by preventing and by strengthening ATP binding to these sites, suggesting that the dynamics of ATP at these sites modulates the enzymes rate. We propose a model by which RecBCD achieves its fast unwinding rate by utilizing the weaker binding sites to increase the flux of ATP to its catalytic sites.

biophysics↗

bioRxiv2017

The mechano-chemistry of reverse transcriptase

Retroviral reverse transcriptase catalyses the synthesis of an integration-competent dsDNA molecule, using as a substrate the viral RNA. Using optical tweezers, we follow the Murine Leukemia Virus reverse transcriptase as it performs strand-displacement polymerization on a template under mechanical force. Our results indicate that reverse transcriptase functions as a Brownian ratchet, with dNTP binding as the rectifying reaction of the ratchet. We also found that reverse transcriptase is a relatively passive enzyme, able to polymerize on structured templates by exploiting their thermal breathing. Finally, our results indicate that the enzyme enters the recently characterized backtracking state from the pre-translocation complex.

biophysics↗

bioRxiv2017

Single-molecule DNA unzipping reveals asymmetric modulation of the transcription factor Egr-1 by its binding site sequence and context

Most functional transcription factor (TF) binding sites deviate from their \"consensus\" recognition motif, although their sites and flanking sequences are often conserved across species. Here, we used single-molecule DNA unzipping with optical tweezers to study how Egr-1, a TF harbouring 3 zinc fingers (ZF1,ZF2 and ZF3), is modulated by the sequence and context of its functional sites in the Lhb gene promoter. We find that both the core 9 base pairs bound to Egr-1 in each of the sites, and the base pairs flanking them, modulate the affinity and structure of the protein-DNA complex. The effect of the flanking sequences is asymmetric, with a stronger effect for the sequence flanking ZF3. Characterization of the dissociation time of Egr-1 revealed that a local, mechanical perturbation of the interactions of ZF3 destabilizes the complex more effectively than a perturbation of the ZF1 interactions. Our results reveal a novel role for ZF3 in the interaction of Egr-1 with other proteins and the DNA, providing insight on the regulation of Lhb and other genes by Egr-1. Moreover, our findings reveal the potential of small changes in DNA sequence to alter transcriptional regulation, and may shed light on the organization of regulatory elements at promoters.

biophysics↗

bioRxiv2017

Mobile nucleosomes and the accessibility of transcription factors to DNA: a modified dynamic equilibrium model

Nucleosomes, the basic building block of chromatin, regulate the accessibility of the transcription machinery to DNA. Recent studies have revealed that the nucleosome's spontaneous, thermally driven positional dynamics are modulated by different factors, and exploited by the cell as a regulatory mechanism. In particular, enrichment of mobile nucleosomes at the promoters of genes suggests that the mobility of nucleosomes may affect the ability of transcription factors to bind DNA. However, a quantitative model describing the effect nucleosome mobility on the effective affinity of transcription factors is lacking. We present here a simple equilibrium model that captures the essence of the effect, and show that modulation of the nucleosome's mobility can be a potent and versatile regulator of transcription factor binding.

biophysics↗