53BP1 Enforces Distinct Pre- and Post-resection Blocks on Homologous Recombination.

53BP1 activity drives genome instability and lethality in BRCA1-deficient mice by inhibiting homologous recombination (HR). The anti-recombinogenic functions of 53BP1 require phosphorylation-dependent interactions with PTIP and RIF1/shieldin effector complexes. While RIF1/shieldin blocks 5'-3' nucleolytic processing of DNA ends, it remains unclear how PTIP antagonizes HR. Here, we show that mutation of the PTIP interaction site in 53BP1 (S25A) allows sufficient DNA2-dependent end resection to rescue the lethality of BRCA1^Δ11 mice, despite increasing RIF1 "end-blocking" at DNA damage sites. However, double-mutant cells fail to complete HR, as excessive shieldin activity also inhibits RNF168-mediated loading of PALB2/RAD51. As a result, BRCA1^Δ1153BP1^S25A mice exhibit hallmark features of HR insufficiency, including premature aging and hypersensitivity to Disruption of shieldin or forced targeting of PALB2 to ssDNA in BRCA1^D1153BP1^S25A cells restores RNF168 recruitment, RAD51 nucleofilament formation, and resistance. Our study therefore reveals a critical function of shieldin post-resection that limits the loading of RAD51.

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