biostudies-literatureDong QMEXT | Japan Society for the Promotion of ScienceMEXT | Japan Society for the Promotion of Science (JSPS)Cancer Research UK5820https://www.ebi.ac.uk/biostudies/studies/S-EPMC12214830biostudies-literatureUnknown16(1)RIF1 is a multifunctional protein that regulates DNA replication and repair. RIF1-deficient cells are hypersensitive to DNA replication stress. Of the two alternatively spliced RIF1 isoforms, called RIF1-Short and RIF1-Long, the RIF1-Long isoform is more capable than RIF1-Short in supporting cell recovery from replication stress. Examining replication stress resistance mechanisms specific to RIF1-Long, we find that prolonged replication stress unexpectedly induces interaction of RIF1-Long with BRCA1. Mechanistically, a phosphorylated SPKF motif unique to the RIF1-Long isoform binds the tandem BRCT domain of BRCA1. BRCA1-RIF1-Long interaction is strongly down-regulated through dephosphorylation by RIF1-associated Protein Phosphatase 1. BRCA1-RIF1-Long interaction requires ATR signaling, and occurs predominantly during S phase. Loss of RIF1-Long impairs the formation of RAD51 foci, and reduces the efficiency of homology-mediated repair at broken replication forks. In summary, our investigation establishes RIF1-Long as a new functional binding partner of the BRCA1-BRCT domain, crucial to protect cells from extended DNA replication stress by enabling RAD51-dependent repair of broken replication forks.Nature communicationsThe human RIF1-Long isoform interacts with BRCA1 to promote recombinational fork repair under DNA replication stress.PMC12214830DRCPGM\100013C1445/A19059JP21H0419JP23H04925C302/A24386Hiraga SIDay MOliver AWSaito YWatts LPPearl LHDonaldson ADParker EDong QKanemaki MTfalseThe human RIF1-Long isoform interacts with BRCA1 to promote recombinational fork repair under DNA replication stress.RIF1 is a multifunctional protein that regulates DNA replication and repair. RIF1-deficient cells are hypersensitive to DNA replication stress. Of the two alternatively spliced RIF1 isoforms, called RIF1-Short and RIF1-Long, the RIF1-Long isoform is more capable than RIF1-Short in supporting cell recovery from replication stress. Examining replication stress resistance mechanisms specific to RIF1-Long, we find that prolonged replication stress unexpectedly induces interaction of RIF1-Long with BRCA1. Mechanistically, a phosphorylated SPKF motif unique to the RIF1-Long isoform binds the tandem BRCT domain of BRCA1. BRCA1-RIF1-Long interaction is strongly down-regulated through dephosphorylation by RIF1-associated Protein Phosphatase 1. BRCA1-RIF1-Long interaction requires ATR signaling, and occurs predominantly during S phase. Loss of RIF1-Long impairs the formation of RAD51 foci, and reduces the efficiency of homology-mediated repair at broken replication forks. In summary, our investigation establishes RIF1-Long as a new functional binding partner of the BRCA1-BRCT domain, crucial to protect cells from extended DNA replication stress by enabling RAD51-dependent repair of broken replication forks.2025-01-01T00:00:00Z2025 Jul2026-06-01T15:31:20.761Z2026-04-08T13:42:20.383ZS-EPMC122148304059549610.1038/s41467-025-60817-y