The tardigrade Damage suppressor protein (Dsup) reduces DNA damage in mammalian cells, yet its mechanism of action within chromatin remains unclear. Here, we investigate how Dsup associates with nuclear chromatin and whether its protective function arises from structural modulation of chromatin, passive shielding of DNA, or modulation of the cellular DNA damage response. Using fluorescence lifetime imaging microscopy of Förster resonance energy transfer (FLIM-FRET), we quantify Dsup–chromatin interactions in live cells and assess their impact on nucleosome organization through a histone FLIM-FRET reporter assay. In parallel, we test whether Dsup reduces double-strand break (DSB) induction by physically buffering DNA, using repair-foci labelling and fluorescence correlation spectroscopy (FCS) of eGFP tracers to probe changes in nuclear accessibility. Finally, to evaluate whether Dsup modulates DNA repair mechanisms, we employ cross-correlation FCS to examine potential interactions between Dsup and key DSB repair proteins under conditions of induced DNA damage. Together, this work aims to delineate the balance between Dsup-mediated DNA protection and mammalian DNA repair dynamics, providing mechanistic insight into how stress-tolerant organisms achieve exceptional genome stability