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The 53BP1-RIF1 pathway antagonizes resection of DNA broken ends and confers PARP inhibitor sensitivity on BRCA1-mutated tumors. However, it is unclear how this pathway suppresses initiation of resection. Here, we identify ASF1 as a partner of RIF1 via an interacting manner similar to its interactions with histone chaperones CAF-1 and HIRA. ASF1 is recruited to distal chromatin flanking DNA breaks by 53BP1-RIF1 and promotes non-homologous end joining (NHEJ) using its histone chaperone activity. Epistasis analysis shows that ASF1 acts in the same NHEJ pathway as RIF1, but via a parallel pathway with the shieldin complex, which suppresses resection after initiation. Moreover, defects in end resection and homologous recombination (HR) in BRCA1- deficient cells are largely suppressed by ASF1 deficiency. Mechanistically, ASF1 compacts adjacent chromatin by heterochromatinization to protect broken DNA ends from BRCA1-mediated resection. Taken together, our findings identified a RIF1-ASF1 histone chaperone complex that promotes changes in high-order chromatin structure to stimulate the NHEJ pathway for DSB repair.

53BP1 with its downstream proteins, RIF1, PTIP and REV7, antagonizes BRCA1-dependent homologous recombination (HR) and promotes non-homologous end joining (NHEJ) in an unclear manner. Here we show that REV7 forms a complex with two proteins, FAM35A and C20ORF196. We demonstrate that FAM35A preferentially binds single-strand DNA (ssDNA) in vitro, and is recruited to DSBs as a complex with C20ORF196 and REV7 downstream of RIF1 in vivo. Epistasis analysis shows that both proteins act in the same pathway as RIF1 in NHEJ. The defects in HR pathway to repair DSBs and the reduction in resection of broken DNA ends in BRCA1-mutant cells can be largely suppressed by inactivating FAM35A or C20ORF196, indicating that FAM35A and C20ORF196 prevent end resection in these cells. Together, our data identified a REV7–FAM35A–C20ORF196 complex that binds and protects broken DNA ends to promote the NHEJ pathway for DSB repair.

Versión postprint próximamente disponible en: http://hdl.handle.net/10261/228202, The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 28 CRISPR/Cas9 screens against 25 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 840 genes whose loss causes either sensitivity or resistance to DNA damaging agents. Mining this dataset, we uncovered that ERCC6L2, which is mutated in a bone-marrow failure syndrome, codes for a canonical non-homologous end-joining pathway factor; that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy., AAQ, GSM and AMH are recipients of long-term EMBO fellowships (ALTF 910-2017, 795-2017 under a CC-BY-NC-ND 4.0 International license. not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available bioRxiv preprint doi: https://doi.org/10.1101/845446; this version posted November 18, 2019. The copyright holder for this preprint (which was 27 and 124-2019, respectively), NH was supported by a Human Frontier Science Program long-term Fellowship, SER is supported by a fellowship from AIRC and SA was a Banting post-doctoral fellow. ASB was supported by a PhD Studentship from AEFAT (Asociación Española Familia Ataxia Telangiectasia) and an EMBO short-term fellowship for a visit to the DD laboratory. The ICRF187 screen in FCL laboratory was funded by grants from the Spanish Government (SAF2017- 89619-R, European Regional Development Fund) and the European Research Council (ERC-CoG2014-647359). Work in the RSW laboratory was supported in part by the US National Institute of Health Intramural Program, US National Institute of Environmental Health Sciences (NIEHS, 1Z01ES102765). DD is a Canada Research Chair (Tier I) and the work was supported from grants from the CIHR (FDN143343 to DD; FRN 123518, PJT-156330 to AM) Canadian Cancer Society grant 705644 (to DD) with additional support to DD from the Krembil Foundation.