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 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 chromatin dynamics are monitored in real time. As expected, PARP1 rapidly accumulates at damage sites, but this response is markedly suppressed upon treatment with Talazoparib, a potent PARP inhibitor. We further observe that laser-induced DNA damage initially localizes to high-density chromatin regions, followed by rapid chromatin relaxation, as indicated by decreasing Hoechst intensity and coefficient of variation (CV). This relaxation is significantly impaired by Talazoparib, consistent with PARP1-dependent chromatin remodeling. Finally, Hoechst-only imaging provides comparable results in both transfected and non-transfected cells, underscoring the robustness of the method and its compatibility with single-label/single-channel acquisition. Chromatin relaxation kinetics were 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.