BRCA1 and 53BP1 Antagonistically Tune a Chromatin Compaction Gradient to Control DNA Break Mobility

The two principal DNA double-strand break (DSB) repair pathways impose fundamentally different mechanical requirements on damaged chromatin. Non-homologous end joining (NHEJ) confines broken DNA ends to preserve their proximity, whereas homologous recombination (HR) requires damaged chromatin to become sufficiently mobile for homology search. While breast cancer associated gene 1 (BRCA1) and p53 binding protein 1 (53BP1) are established molecular regulators of DSB repair pathway choice, the chromatin architecture that translates this choice into the required mechanical state of the break has remained undefined. Here, we developed a multiplexed live-cell imaging framework combining phasor-based fluorescence lifetime imaging microscopy of histone Förster resonance energy transfer (FLIM-FRET) with single-particle tracking (SPT) of fluorescently labelled DSB repair proteins, enabling nanoscale chromatin compaction to be directly correlated with the mobility of individual DSBs in living cells. We show that DSB induction generates a chromatin compaction gradient between locally de-compacted repair sites and the surrounding compact chromatin. This gradient regulates DSB mobility, with HR-associated breaks exhibiting steeper gradients, increased mobility and greater exploration of the nuclear space than NHEJ-associated breaks, which remain spatially confined. BRCA1 and 53BP1 antagonistically regulate this gradient: BRCA1 promotes chromatin opening and restrains DSB mobility, whereas 53BP1 drives compaction and enhances movement. Loss of BRCA1 collapses this balance, causing chromatin over-compaction and DSB hyper-mobility that collectively drive poly(ADP-ribose) polymerase (PARP) inhibitor-induced chromosome mis-joining, a phenotype rescued by 53BP1 depletion. Together, our findings identify the chromatin compaction gradient as a biophysical regulator of DSB mobility that fulfils the distinct mechanical requirements of HR and NHEJ, providing a mechanistic basis for PARP inhibitor sensitivity and resistance in BRCA1-deficient cancers.