Unraveling the landscape of factors promoting DNA repair by Non-Homologous End-Joining

DNA double-strand break (DSBs) are highly cytotoxic DNA lesions which can be repaired by two major pathways: non-homologous end-joining (NHEJ) and homologous recombination. Proper DNA repair pathway choice is governed by the opposing activities of 53BP1 and BRCA1 in a cell cycle-dependent manner. 53BP1, in complex with his effectors RIF1 and REV7, stimulates NHEJ and antagonizes both the loading of BRCA1 at DSBs and DNA end resection in G1 phase of the cell cycle. However, it remains unknown how the adaptor protein REV7 promotes NHEJ and limits DNA end resection.

Therefore, we performed a systematic proteomic analysis of REV7 composed of (i)a standard affinity purification coupled to mass spectrometry (AP/MS); (ii)a proximity-based biotin labeling approach (BioID). We identified 140 high-confidence REV7 interactors that were common to both approaches and restricted our subsequent validation to 11 candidates that have been previously reported in other proteomic profiling of REV7. Using several GFP-based DNA reporter assays, we identified FAM35 as a novel and critical component of the NHEJ pathway and further investigated its contribution to this pathway.

First, we confirmed that FAM35 interacts with REV7 by co-immunoprecipitation. Interestingly, we observed that FAM35 accumulate at DSBs in a 53BP1-, RIF1 and REV7-dependent manner, placing FAM35A as a downstream effector of REV7. As anticipated, depletion of FAM35 impairs NHEJ-mediated DNA repair and results in a hypersensitivity to ionizing radiation. Remarkably, reduction of FAM35A levels also compromises antibody diversification by class switch recombination in B-cells. Using a genome-wide CRISPR/Cas9-based approach, we identified C20orf196 as a partner of FAM35A in the NHEJ pathway. We confirmed that C20orf196 co-immunoprecipitates with FAM35A. Similar to what we observed with FAM35, depletion of C20orf196 impairs NHEJ in a DNA repair reporter assay and class switch recombination in B-cells, suggesting that both factors co-operate to promote NHEJ downstream of REV7.

To better define the clinical relevance of FAM35A, we interrogated publicly available patient database. Strikingly, high levels of FAM35A correlated with a poor prognosis for patients affected by Basal/Triple Negative Breast Cancer (TNBC). Together, we establish for the first time FAM35A as novel effector of REV7 in promoting NHEJ-mediated DNA repair and as a biomarker for a subset of BC patients.