bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.10.01.679728

Tracking DNA damage localization and chromatin remodeling in live cells using time-resolved quantitative analysis of DNA counterstains

Paterno, G.·Longo, E.·Lanzano, L.
View original source ↗Export metadata ↓

Abstract

DNA damage profoundly impacts genome stability and cellular homeostasis, and its repair is tightly coordinated with local chromatin remodeling. However, monitoring these rapid chromatin changes in living cells remains challenging. We recently developed QUANDO, an imaging-based method that exploits a simple DNA counterstain to investigate the subnuclear localization of DNA damage in fixed cells. Here, we adapt this approach to track chromatin remodeling at laser-induced DNA damage sites in live cells using Hoechst-based staining. Specifically, PARP1-expressing HeLa cells are exposed to UV laser micro-irradiation in a defined nuclear region, and PARP1 and chromatin dynamics are monitored in real time. We observe that PARP1 rapidly accumulates in the irradiated region but with a heterogeneous pattern: PARP1 initially localizes to high-density chromatin regions (where the concentration of the sensitizer is higher) and gradually redistributes over the whole irradiated region. At the same time, we observe rapid chromatin relaxation, as indicated by decreasing Hoechst intensity and coefficient of variation (CV). In this framework, the PARP inhibitor Talazoparib has the following effects: it slows down PARP1 accumulation, it freezes the PARP1 heterogeneous pattern and blocks chromatin relaxation. Finally, we show that Hoechst-only imaging is sufficient to observe chromatin remodeling: measured chromatin relaxation kinetics are similar in transfected and non-transfected cells, confirming that staining with Hoechst is sufficient for studying chromatin dynamics bypassing the complexities of transfection. These findings underscore the dynamic interplay between DNA damage and chromatin remodeling, demonstrating how conventional nuclear counterstaining can reveal rapid chromatin changes at damage sites, offering new perspectives for investigating genome stability in live cells.

Source connections

Explore related subjects

biophysicsPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Paterno, G., Longo, E., Lanzano, L.. 2025-10-02. Tracking DNA damage localization and chromatin remodeling in live cells using time-resolved quantitative analysis of DNA counterstains. https://doi.org/10.1101/2025.10.01.679728

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Design of colipids with controlled lateral distance of head group moieties for targeting strategies in the field of lipid-based nanoparticles

Lipid nanoparticles and liposomes are nanoparticle drug formulations which are firmly established in clinics. In order to expand their application and improve efficacy, surface modifications can be a key strategy, especially for cell specific targeting. Targeting can be achieved by integrating ligand functionalized lipids or lipidoids on the nanoparticle surface. In this case, not solely the targeting ligand itself, but also the structure of the whole targeting lipid/lipidoid can affect the interaction of the targeting ligand with the binding motive on cells. Especially, distal spacer structures between lipid anchor and ligand unit, often composed of polyethylene glycol chains, provide a certain distance of the ligand from the nanoparticle surface. A concept which is currently neglected is the control of the lateral distance between ligand structures on the nanoparticle. Herein we investigated the possibility to design lipidoids with a lateral spacer composed of polyethylene glycol units. We synthesized and characterized different lipidoids which provide a primary amine in the head group for possible ligand coupling. In our model lipidoids the head groups were functionalized with a paramagnetic probe to apply electron paramagnetic resonance (EPR) methods for the characterization of the lipidoid interaction with model nanoparticles, in our case liposomes. Continuous wave EPR was used to study the efficiency of the novel lipidoids to integrate into lipid bilayers of model liposomes, which was shown to depend on lipidoid structure and liposome composition. Furthermore, using double electron-electron resonance (DEER), we observed lipidoid conformational changes occurring upon integration into liposomes.

biophysics↗
bioRxiv2026

Beyond Sequence and Structure: Pretraining PLMs on Continuous Biophysical Fields

We introduce the Biophysical Transformer, a protein language model that adds continuous biophysical fields as a third pretraining signal beside sequence and structure. A Poisson-Boltzmann solver computes the electrostatic potential around each three-residue token, and the model learns to predict it from sequence alone. A rotationally invariant spherical-harmonic descriptor serves as the loss target, eliminating the need for frame alignment during training and for coordinates at inference. Against a matched, one-flag control on the FLIP Meltome thermostability benchmark, the field objective raises Spearman correlation on every trained head of both human splits.The gain on human survives residualizing dipeptide composition under two architecturally different readouts and survives removing the test proteins that overlap the pretraining corpus. No effect survives on the organism-mixed split. At 1.1M parameters, both the Biophysical Transformer and its sequence-only control exceed FLIP's one-hot CNN and untrained-ESM baselines on every split. All results rest on a single pretraining run per arm,and the current design does not separate field content from generic regularization.

biophysics↗
bioRxiv2026

An auditable harness for biomolecular dynamics simulations by AI agents

AI agents now generate biological hypotheses faster than they can be tested. Hypotheses stated at the atomic level can be examined with molecular dynamics (MD) simulations. For new questions without a reference, trust in an agent-run simulation rests on a tool-written process record, not the agent's account. Existing MD agents leave this record to the language model. Here we present MDClaw, a harness whose validating tools and directed acyclic graph (DAG) check stages before execution and record every simulation stage. On MDDataBench, which grades outputs against deposited simulations, four open-weight models with MDClaw passed all three runs on 81-100% of 31 test systems. The DAG complements this outcome check. An ablation without the DAG revealed 11 successful runs that had skipped minimization or equilibration, or simulated a different structure. MDClaw carried questions from human and AI scientists through simulation to trajectories traceable to their inputs and settings.

biophysics↗