The BRCT domain is a modular platform for signal transduction in the DNA damage response

Protein modular domains are critical for the cellular response to intra- and extracellular signaling, facilitating the assembly of dynamic multi-protein complexes in response to specific stimuli. The BRCA1 C-Terminal (BRCT) domain is present in 23 proteins in the human proteome, containing 46 individual BRCT modules organized as 14 singletons, 16 tandems, and one quartet of BRCT functional units. The BRCT singleton consists of a central core of four parallel beta sheets flanked by two alpha helices on one side and one alpha helix on the opposite side. The BRCT domain is a versatile protein-modular domain that has emerged as an integrator of signals in maintaining genomic integrity. We previously charted the protein-protein interaction network of seven tandem BRCT domains (tBRCT), revealing previously unknown components in DNA damage signaling. Here, we present a comprehensive BRCT-centered human protein-protein interaction network (PPN) comprising 46 BRCT domains organized into various domain architectures, building on our previous work. This network was determined by integrating data from literature curation, yeast two-hybrid (Y2H) screens, and tandem affinity purification coupled to mass spectrometry (TAP-MS) and comprises 3,162 highly curated BRCT-dependent interactions and 1,672 unique human proteins. The BRCT-containing protein PAXIP1 (also known as PTIP) is critical for DNA double-strand break repair by non-homologous end joining. PAXIP1 has a distinctive structural organization, containing three tBRCT domains, which present a unique set of PPN. Interestingly, the last four BRCT domains form a unique quartet (qBRCT) in the human proteome, binding to a set of proteins not observed in either of the tBRCTs alone. These data indicate that distinct interacting partners may be recruited by different combinations of higher-order BRCT domain organization to assemble inter- or intramolecular complexes, accommodating combinatorial interactions in response to genotoxic stimuli. Moreover, we mapped 62 unique phosphorylation sites at 19 BRCT domains. We identified that PAXIP1 qBRCT phosphorylation alters its binding to gH2AX, providing an additional layer of regulation by modulating BRCT-mediated ligand recognition. The BRCT PPN resource charted here provides a comprehensive analysis of these modular protein domains in the human proteome, contributing to an understanding of cancer and its response to genotoxic clinical therapies.