RF-SIRF reveals a replication stress-specific epigenetic code by spatio-temporal mapping of reversed forks.

DNA replication stress responses are guardians of genomic stability critical during development, hematopoiesis, cancer therapy response, aging and disease suppression. Central to these responses are reversed forks (RF), which are distinct four-way DNA structures formed during DNA replication stalling to protect against toxic DNA lesions. Historically, RF detection relies on specialized electron microscopy, precluding studies within their native cellular context. By harnessing intrinsic bio-physical properties of RFs, we here present a quantitative method to map RFs with single-cell resolution (RF-SIRF). RF-SIRF reveals that RFs accumulate at the nuclear periphery during early-mid S-phase of the cell cycle. Crucially, RFs possess a specialized chromatin landscape and utilize an epigenetic replication stress code distinct from transcription, explaining the selective recruitment of DNA stress response proteins to RFs. Collectively, RF-SIRF enables robust quantitative, temporal, spatial and proteomic analyses of reversed forks, empowering advanced cellular and medical investigations of DNA replication stress responses.