DNA double-strand break (DSB) repair pathway choice is governed by the opposing activities of 53BP1 and BRCA1. 53BP1 stimulates nonhomologous end joining (NHEJ), whereas BRCA1 promotes end resection and homologous recombination (HR). Here we show that 53BP1 is an inhibitor of BRCA1 accumulation at DSB sites, specifically in the G1 phase of the cell cycle. ATM-dependent phosphorylation of 53BP1 physically recruits RIF1 to DSB sites, and we identify RIF1 as the critical effector of 53BP1 during DSB repair. Remarkably, RIF1 accumulation at DSB sites is strongly antagonized by BRCA1 and its interacting partner CtIP. Lastly, we show that depletion of RIF1 is able to restore end resection and RAD51 loading in BRCA1-depleted cells. This work therefore identifies a cell cycle-regulated circuit, underpinned by RIF1 and BRCA1, that governs DSB repair pathway choice to ensure that NHEJ dominates in G1 and HR is favored from S phase onward.

53BP1 (TP53BP1) is a chromatin-associated factor that promotes immunoglobulin class switching and DNA double-strand break (DSB) repair by non-homologous end joining. To accomplish its function in DNA repair, 53BP1 accumulates at DSB sites downstream of the RNF168 ubiquitin ligase. How ubiquitin recruits 53BP1 to break sites remains enigmatic since its relocalization involves recognition of H4 Lys20 (H4K20) methylation by its Tudor domain. Here we elucidate how 53BP1 is recruited to the chromatin that flanks DSB sites. We show that 53BP1 recognizes mono-nucleosomes containing dimethylated H4K20 (H4K20me2) and H2A ubiquitylated on Lys15 (H2AK15ub), the latter being a product of RNF168 action on chromatin. 53BP1 binds to nucleosomes minimally as a dimer using its previously characterized methyl-lysine-binding Tudor domain and a C-terminal extension, termed the ubiquitylation-dependent recruitment (UDR) motif, which interacts with the epitope formed by H2AK15ub and its surrounding residues on the H2A tail. 53BP1 is therefore a bivalent histone modification reader that recognizes a histone “code” produced by DSB signaling.

Biallelic inactivation of BRCA1 or BRCA2 is associated with a pattern of genome-wide mutations known as signature 3. By analyzing ∼1,000 breast cancer samples, we confirmed this association and established that germline nonsense and frameshift variants in PALB2, but not in ATM or CHEK2, can also give rise to the same signature. We were able to accurately classify missense BRCA1 or BRCA2 variants known to impair homologous recombination (HR) on the basis of this signature. Finally, we show that epigenetic silencing of RAD51C and BRCA1 by promoter methylation is strongly associated with signature 3 and, in our data set, was highly enriched in basal-like breast cancers in young individuals of African descent.