ABSTRACT
The response to DNA double-strand breaks (DSBs) entails the hierarchical recruitment of proteins orchestrated by ATM-dependent phosphorylation and RNF8-mediated chromatin ubiquitylation. As in most ubiquitin-dependent processes, the ordered accumulation of DNA repair factors at the break site relies on ubiquitin-binding domains (UBDs). However, how UBDs select their ligands is poorly understood, and therefore we sought to uncover the basis for selectivity in the ubiquitin-dependent DSB response. We show that RNF168, its paralog RNF169, RAD18, and the BRCA1-interacting RAP80 protein accumulate at DSB sites through the use of bipartite modules composed of UBDs juxtaposed to peptide motifs that provide specificity. These sequences, named LR motifs (LRMs), are transferable, and we show that the RNF169 LRM2 binds to nucleosomes, the substrates of RNF168. The LRM-based selection of ligands is a parsimonious means to build a highly discrete ubiquitin-based signaling pathway such as the DNA damage response.
Subject(s)
Carrier Proteins/metabolism , DNA Breaks, Double-Stranded , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Carrier Proteins/chemistry , Carrier Proteins/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , HCT116 Cells , HEK293 Cells , Histone Chaperones , Humans , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Phosphorylation/physiology , Ubiquitin/physiology , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics , Ubiquitination/physiologyABSTRACT
Ubiquitylation entails the concerted action of E1, E2, and E3 enzymes. We recently reported that OTUB1, a deubiquitylase, inhibits the DNA damage response independently of its isopeptidase activity. OTUB1 does so by blocking ubiquitin transfer by UBC13, the cognate E2 enzyme for RNF168. OTUB1 also inhibits E2s of the UBE2D and UBE2E families. Here we elucidate the structural mechanism by which OTUB1 binds E2s to inhibit ubiquitin transfer. OTUB1 recognizes ubiquitin-charged E2s through contacts with both donor ubiquitin and the E2 enzyme. Surprisingly, free ubiquitin associates with the canonical distal ubiquitin-binding site on OTUB1 to promote formation of the inhibited E2 complex. Lys48 of donor ubiquitin lies near the OTUB1 catalytic site and the C terminus of free ubiquitin, a configuration that mimics the products of Lys48-linked ubiquitin chain cleavage. OTUB1 therefore co-opts Lys48-linked ubiquitin chain recognition to suppress ubiquitin conjugation and the DNA damage response.
Subject(s)
Cysteine Endopeptidases/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitinated Proteins/metabolism , Amino Acid Substitution , Cell Line , Crystallography, X-Ray , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/genetics , Deubiquitinating Enzymes , Humans , Kinetics , Models, Molecular , Mutagenesis, Site-Directed , Organisms, Genetically Modified , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Quaternary , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Ubiquitin/chemistry , Ubiquitin/metabolism , Ubiquitin-Conjugating Enzymes/chemistry , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitination , Yeasts/genetics , Yeasts/growth & developmentABSTRACT
53BP1 (also called TP53BP1) is a chromatin-associated factor that promotes immunoglobulin class switching and DNA double-strand-break (DSB) repair by non-homologous end joining. To accomplish its function in DNA repair, 53BP1 accumulates at DSB sites downstream of the RNF168 ubiquitin ligase. How ubiquitin recruits 53BP1 to break sites remains unknown as its relocalization involves recognition of histone H4 Lys 20 (H4K20) methylation by its Tudor domain. Here we elucidate how vertebrate 53BP1 is recruited to the chromatin that flanks DSB sites. We show that 53BP1 recognizes mononucleosomes containing dimethylated H4K20 (H4K20me2) and H2A ubiquitinated on Lys 15 (H2AK15ub), the latter being a product of RNF168 action on chromatin. 53BP1 binds to nucleosomes minimally as a dimer using its previously characterized methyl-lysine-binding Tudor domain and a carboxy-terminal extension, termed the ubiquitination-dependent recruitment (UDR) motif, which interacts with the epitope formed by H2AK15ub and its surrounding residues on the H2A tail. 53BP1 is therefore a bivalent histone modification reader that recognizes a histone 'code' produced by DSB signalling.
Subject(s)
DNA Damage , Histones/chemistry , Histones/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Lysine/metabolism , Ubiquitin/metabolism , Ubiquitination , Amino Acid Motifs , Amino Acid Sequence , Animals , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Cell Line , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/deficiency , Chromosomal Proteins, Non-Histone/genetics , DNA Breaks, Double-Stranded , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Female , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/deficiency , Intracellular Signaling Peptides and Proteins/genetics , Male , Mice , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Nucleosomes/chemistry , Nucleosomes/metabolism , Protein Binding , Protein Structure, Tertiary , Schizosaccharomyces , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces pombe Proteins/metabolism , Signal Transduction , Tumor Suppressor p53-Binding Protein 1ABSTRACT
The repair of DNA double strand breaks by homologous recombination relies on the unique topology of the chains formed by Lys-63 ubiquitylation of chromatin to recruit repair factors such as breast cancer 1 (BRCA1) to sites of DNA damage. The human RING finger (RNF) E3 ubiquitin ligases, RNF8 and RNF168, with the E2 ubiquitin-conjugating complex Ubc13/Mms2, perform the majority of Lys-63 ubiquitylation in homologous recombination. Here, we show that RNF8 dimerizes and binds to Ubc13/Mms2, thereby stimulating formation of Lys-63 ubiquitin chains, whereas the related RNF168 RING domain is a monomer and does not catalyze Lys-63 polyubiquitylation. The crystal structure of the RNF8/Ubc13/Mms2 ternary complex reveals the structural basis for the interaction between Ubc13 and the RNF8 RING and that an extended RNF8 coiled-coil is responsible for its dimerization. Mutations that disrupt the RNF8/Ubc13 binding surfaces, or that truncate the RNF8 coiled-coil, reduce RNF8-catalyzed ubiquitylation. These findings support the hypothesis that RNF8 is responsible for the initiation of Lys-63-linked ubiquitylation in the DNA damage response, which is subsequently amplified by RNF168.