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

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.