Abstract
DNA replication in mammalian cells proceeds in chromosomal domains during defined windows of S phase; however, the mechanisms coordinating replication with restoration of chromatin states remain incompletely understood. Here, we identify p53-binding protein 1 (53BP1) condensates as regulators of heterochromatin replication and epigenetic restoration. Loss of 53BP1 delays mid-to-late S-phase progression, compromises recruitment of epigenetic restoration factors, and impairs timely completion of heterochromatin replication and restoration of heterochromatin-associated epigenetic marks. These defects were rescued by wild-type or condensate-competent 53BP1, but not by condensate-deficient mutants. Further, 53BP1 condensate formation and its role in heterochromatin replication depend on interaction with proliferating cell nuclear antigen, which also mediates 53BP1 relocalization from condensates to DNA damage foci upon genotoxic stress. Together, our findings reveal a previously unrecognized role for 53BP1 condensates in coordinating heterochromatin replication and epigenetic restoration, while serving as a reservoir for DNA damage response.