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The BRCA1 tumour suppressor and its heterodimeric partner BARD1 constitute an E3‐ubiquitin (Ub) ligase and function in DNA repair by unknown mechanisms. We show here that the Caenorhabditis elegans BRCA1/BARD1 (CeBCD) complex possesses an E3‐Ub ligase responsible for ubiquitylation at DNA damage sites following ionizing radiation (IR). The DNA damage checkpoint promotes the association of the CeBCD complex with E2‐Ub conjugating enzyme, Ubc5(LET‐70), leading to the formation of an active E3‐Ub ligase on chromatin following IR. Correspondingly, defects in Ubc5(let‐70) or the DNA damage checkpoint genes atl‐ 1 or mre‐11 abolish CeBCD‐dependent ubiquitylation in vivo . Extending these findings to human cells reveals a requirement for UbcH5c, the MRN complex, γ‐H2AX and a co‐dependence for ATM and ATR kinases for BRCA1‐dependent ubiquitylation at DNA damage sites. Furthermore, we demonstrate that the DNA damage checkpoint promotes the association between BRCA1 and UbcH5c to form an active E3‐Ub ligase on chromatin after IR. These data reveal that BRCA1‐dependent ubiquitylation is activated at sites of DNA repair by the checkpoint as part of a conserved DNA damage response.

This paper reports an approach to measure equilibrium binding affinities for interacting proteins in high throughput, allowing the rapid and quantitative profiling of the specificity of interaction motifs. Many protein interactions are mediated by small linear motifs interacting specifically with defined families of globular domains. Quantifying the specificity of a motif requires measuring and comparing its binding affinities to all its putative target domains. To this end, we developed the high-throughput holdup assay, a chromatographic approach that can measure up to 1,000 domain-motif equilibrium binding affinities per day. After benchmarking the approach on 210 PDZ-peptide pairs with known affinities, we determined the affinities of two viral PDZ-binding motifs derived from human papillomavirus E6 oncoproteins for 209 PDZ domains covering 79% of the human 'PDZome'. We obtained sharply sequence-dependent binding profiles that quantitatively describe the PDZome recognition specificity of each motif. This approach, applicable to many categories of domain-ligand interactions, has wide potential for quantifying the specificities of interactomes.

Protein–protein interactions organize the localization, clustering, signal transduction, and degradation of cellular proteins and are therefore implicated in numerous biological functions. These interactions are mediated by specialized domains able to bind to modified or unmodified peptides present in binding partners. Among the most broadly distributed protein interaction domains, PSD95-disc large-zonula occludens (PDZ) domains are usually able to bind carboxy-terminal sequences of their partners. In an effort to accelerate the discovery of PDZ domain interactions, we have constructed an array displaying 96% of the human PDZ domains that is amenable to rapid two-hybrid screens in yeast. We have demonstrated that this array can efficiently identify interactions using carboxy-terminal sequences of PDZ domain binders such as the E6 oncoviral protein and protein kinases (PDGFRβ, BRSK2, PCTK1, ACVR2B, and HER4); this has been validated via mass spectrometry analysis. Taking advantage of this array, we show that PDZ domains of Scrib and SNX27 bind to the carboxy-terminal region of the planar cell polarity receptor Vangl2. We also have demonstrated the requirement of Scrib for the promigratory function of Vangl2 and described the morphogenetic function of SNX27 in the early Xenopus embryo. The resource presented here is thus adapted for the screen of PDZ interactors and, furthermore, should facilitate the understanding of PDZ-mediated functions.