(2007). RINN3 (C20ORF196). Recruitment of shieldin to DSBs, via the ATM-RNF8-RNF168C53BP1-RIF1 axis, promotes NHEJ-dependent repair of intrachromosomal breaks, immunoglobulin class-switch recombination (CSR), and fusion of unpro-tected telomeres. Shieldin functions as a downstream effector of 53BP1-RIF1 in restraining DNA end resec-tion and in sensitizing provides synthetic viability to (hereafter APEX) at the N terminus of the endogenous in U2OS cells (Figure 1A). The FLAG tag was included for affinity purification and MS (AP-MS) of the bait proteins, and APEX was included for biotinylation-based analysis of bait-proximal networks (hereafter PROX-NET). Correctly modified cell clones were identified by genomic PCR and immunoblotting (Figures 1B and S1A), and competent localization of the bait proteins to DSBs was confirmed by analyzing their localization to ionization radiation-induced foci (IRIF) (Figure S1B). The functionality of APEX was confirmed by selective biotinylation in the engineered cells (Figure 1C). Open in a separate window Figure 1. PROX-NET Analyses of the Endogenous 53BP1, BRCA1, and MDC1(A) Strategy for CRISPR-based 33-FLAG-APEX2 tagging, exemplified by picture of the neighborhood interaction networks. We used SILAC ratios of interactors, their bait-specificity, and enrichment frequency in replicate experiments to calculate pairwise correlation for all proteins that were enriched in our dataset and used these data to generate interaction networks (Figure S3). These networks faithfully recapitulated many known binary interactions and protein complexes, including the BRCA1 A, MRN, G6PD activator AG1 CFIm, and shelterin, highlighting the potential of our approach in constructing interaction networks. Identification of RINN1 To further demonstrate the potential of our datasets in identifying novel components of the DDR, we were intrigued to find a novel 250-amino-acid protein (CTC-534A2.2; UniProt: “type”:”entrez-protein”,”attrs”:”text”:”Q6ZNX1″,”term_id”:”74710788″,”term_text”:”Q6ZNX1″Q6ZNX1) that was among the top hits that was reproducibly enriched in proximity to 53BP1, but not with BRCA1 or MDC1 (Figure 3A). In our networks, it was predicted to interact with proteins including USP28 and REV7, which function with 53BP1 in the DSB repair (Boersma et al., 2015; Xu et al., 2015). We named this novel protein as RINN1 (REV7-interacting novel NHEJ regulator 1) based on its function that will become apparent later. is encoded by a single exon, and the gene is nested within the first intron of (Figure S4A). We generated a RINN1 antibody (Figure S4B) and confirmed RINN1 expression in diverse human cell lines (Figure S4C). Open in a separate window Figure 3. RINN1 Directly Interacts with REV7(A) The bar G6PD activator AG1 chart shows log2 fold enrichment of 53BP1, RIF1, G6PD activator AG1 and RINN1 in PROX-NET dataset of 53BP1, BRCA1, and MDC1. The network shows predicted interactions of RINN1. (B) Reciprocal interaction between RINN1 and REV7. FLAG-RINN1 and GFP-REV7 were immunoprecipitated, and interaction with the endogenous REV7 and RINN1, respectively, was analyzed by immunoblotting. (C) Mapping of REV7 Hpt interacting region in RINN1. (D) The elution profiles (the top panel) G6PD activator AG1 of recombinant His6-REV7R124A and RINN128C83 and His6-REV7RINN128C83 in a S75 10/300 analytical size exclusion chromatography (SEC) column. Fractions from analytical SEC runs were analyzed by SDS-PAGE and Coomassie staining for the whole elution range, showing elution of the REV7RINN128C83 complex. (E) Thermodynamic analysis of the REV7R124A-RINN128C83 interaction (left panel). REV7R124A binds tightly to RINN128C83 with a nanomolar affinity (KD = 15.8 nM). The control (the right panel) shows titration of RINN128C83 in buffer alone. (F) Mutation of conserved P53 and P58 abolishes RINN1 interaction with REV7. See also Figure S4 and Table S4. RINN1 Directly Interacts with REV7 To identify its function, we performed label-free AP-MS analyses of RINN1 and identified REV7 as an interactor; conversely, RINN1 was enriched in REV7 pull-downs (Table S4). RINN1 and REV7 interacted reciprocally, the interaction was unaffected by DNA damage, and the interaction region was mapped to amino acids 28C83 in RINN1 (Figures 3B and ?and3C).3C). Recombinant RINN128C83 and REV7 formed a complex (Figure 3D), and the interaction affinity was measured to be ~15 nM, demonstrating a direct interaction between these proteins (Figures 3E and S4D). Sequence alignment of the REV7-interacting region in RINN1 revealed four residues (F38, W41, P53, and P58) that are invariably conserved in diverse species (Figure S4E). Mutation of bulky Trp and Phe could affect the overall protein structure; therefore, we mutated the conserved proline residues, which are mostly found in loops and thus G6PD activator AG1 are less likely to impact the overall protein structure. Notably, the mutant RINN1P53A,P58A was completely deficient for interaction with REV7 (Figure 3F). RINN1 Functions Downstream of 53BP1-RIF1 Based on its novelty and strong interaction with REV7, we investigated the function of RINN1.