Molecular Basis for Phosphorylation-dependent SUMO Recognition by the DNA Repair Protein RAP80

Recognition and repair of double-stranded DNA breaks (DSB) involves the targeted recruitment of BRCA tumor suppressors to damage foci through binding of both ubiquitin (Ub) and the Ub-like modifier SUMO. RAP80 is a component of the BRCA1 A complex, and plays a key role in the recruitment process through the binding of Lys⁶³-linked poly-Ub chains by tandem Ub interacting motifs (UIM). RAP80 also contains a SUMO interacting motif (SIM) just upstream of the tandem UIMs that has been shown to specifically bind the SUMO-2 isoform. The RAP80 tandem UIMs and SIM function collectively for optimal recruitment of BRCA1 to DSBs, although the molecular basis of this process is not well understood. Using NMR spectroscopy, we demonstrate that the RAP80 SIM binds SUMO-2, and that both specificity and affinity are enhanced through phosphorylation of the canonical CK2 site within the SIM. The affinity increase results from an enhancement of electrostatic interactions between the phosphoserines of RAP80 and the SIM recognition module within SUMO-2. The NMR structure of the SUMO-2·phospho-RAP80 complex reveals that the molecular basis for SUMO-2 specificity is due to isoform-specific sequence differences in electrostatic SIM recognition modules.     

Molecular basis of Lys-63-linked polyubiquitination inhibition by the interaction between human deubiquitinating enzyme OTUB1 and ubiquitin-conjugating enzyme UBC13

UBC13 is the only known E2 ubiquitin (Ub)-conjugating enzyme that produces Lys-63-linked Ub chain with its cofactor E2 variant UEV1a or MMS2. Lys-63-linked ubiquitination is crucial for recruitment of DNA repair and damage response molecules to sites of DNA double-strand breaks (DSBs). A deubiquitinating enzyme OTUB1 suppresses Lys-63-linked ubiquitination of chromatin surrounding DSBs by binding UBC13 to inhibit its E2 activity independently of the isopeptidase activity. OTUB1 strongly suppresses UBC13-dependent Lys-63-linked tri-Ub production, whereas it allows di-Ub production in vitro. The mechanism of this non-canonical OTUB1-mediated inhibition of ubiquitination remains to be elucidated. Furthermore, the atomic level information of the interaction between human OTUB1 and UBC13 has not been reported. Here, we determined the crystal structure of human OTUB1 in complex with human UBC13 and MMS2 at 3.15 Å resolution. The presented atomic-level interactions were confirmed by surface-plasmon resonance spectroscopy with structure-based mutagenesis. The designed OTUB1 mutants cannot inhibit Lys-63-linked Ub chain formation in vitro and histone ubiquitination and 53BP1 assembly around DSB sites in vivo. Finally, we propose a model for how capping of di-Ub by the OTUB1-UBC13-MMS2/UEV1a complex efficiently inhibits Lys-63-linked tri-Ub formation.     

RAP80 protein is important for genomic stability and is required for stabilizing BRCA1-A complex at DNA damage sites in vivo

RAP80 (receptor-associated protein 80) is a ubiquitin-binding protein that can specifically recognize and bind to Lys-63-linked polyubiquitin chains, thus targeting the BRCA1-A complex to DNA damage sites. To study the role of RAP80 in vivo, we generated RAP80-deficient mice. The deficient mice are prone to B-cell lymphomagenesis. B-cell lymphomas in RAP80-deficient mice are nearly diploid but harbor clonal chromosome translocations. Moreover, the deficient mice are hypersensitive to ionizing radiation. Repair of ionizing radiation-induced DNA double-strand breaks is impaired in RAP80-deficient mouse embryonic fibroblasts. Mechanistically, loss of RAP80 suppresses recruitment of the BRCA1-A complex to DNA damage sites and abrogates the DNA damage repair process at DNA damage sites. Taken together, these results reveal that RAP80 plays a crucial role in the DNA damage response and in maintaining genomic integrity.     

Structural basis for specific recognition of Lys 63‐linked polyubiquitin chains by tandem UIMs of RAP80

RAP80 has a key role in the recruitment of the Abraxas–BRCC36–BRCA1–BARD1 complex to DNA‐damage foci for DNA repair through specific recognition of Lys 63‐linked polyubiquitinated proteins by its tandem ubiquitin‐interacting motifs (UIMs). Here, we report the crystal structure of the RAP80 tandem UIMs (RAP80‐UIM1‐UIM2) in complex with Lys 63‐linked di‐ubiquitin at 2.2 Å resolution. The two UIMs, UIM1 and UIM2, and the α‐helical inter‐UIM region together form a continuous 60 Å‐long α‐helix. UIM1 and UIM2 bind to the proximal and distal ubiquitin moieties, respectively. Both UIM1 and UIM2 of RAP80 recognize an Ile 44‐centered hydrophobic patch on ubiquitin but neither UIM interacts with the Lys 63‐linked isopeptide bond. Our structure suggests that the inter‐UIM region forms a 12 Å‐long α‐helix that ensures that the UIMs are arranged to enable specific binding of Lys 63‐linked di‐ubiquitin. This was confirmed by pull‐down analyses using RAP80‐UIM1‐UIM2 mutants of various length inter‐UIM regions. Further, we show that the Epsin1 tandem UIM, which has an inter‐UIM region similar to that of RAP80‐UIM1‐UIM2, also selectively binds Lys 63‐linked di‐ubiquitin.     

The ubiquitin-selective segregase VCP/p97 orchestrates the response to DNA double-strand breaks

Unrepaired DNA double-strand breaks (DSBs) cause genetic instability that leads to malignant transformation or cell death. Cells respond to DSBs with the ordered recruitment of signalling and repair proteins to the site of lesion. Protein modification with ubiquitin is crucial for the signalling cascade, but how ubiquitylation coordinates the dynamic assembly of these complexes is poorly understood. Here, we show that the human ubiquitin-selective protein segregase p97 (also known as VCP; valosin-containing protein) cooperates with the ubiquitin ligase RNF8 to orchestrate assembly of signalling complexes and efficient DSB repair after exposure to ionizing radiation. p97 is recruited to DNA lesions by its ubiquitin adaptor UFD1-NPL4 and Lys-48-linked ubiquitin (K48-Ub) chains, whose formation is regulated by RNF8. p97 subsequently removes K48-Ub conjugates from sites of DNA damage to orchestrate proper association of 53BP1, BRCA1 and RAD51, three factors critical for DNA repair and genome surveillance mechanisms. Impairment of p97 activity decreases the level of DSB repair and cell survival after exposure to ionizing radiation. These findings identify the p97-UFD1-NPL4 complex as an essential factor in ubiquitin-governed DNA-damage response, highlighting its importance in guarding genome stability.     

S5a promotes protein degradation by blocking synthesis of nondegradable forked ubiquitin chains

Ubiquitin (Ub)–protein conjugates formed by purified ring‐finger or U‐box E3s with the E2, UbcH5, resist degradation and disassembly by 26S proteasomes. These chains contain multiple types of Ub forks in which two Ub's are linked to adjacent lysines on the proximal Ub. We tested whether cells contain factors that prevent formation of nondegradable conjugates and whether the forked chains prevent proteasomal degradation. S5a is a ubiquitin interacting motif (UIM) protein present in the cytosol and in the 26S proteasome. Addition of S5a or a GST‐fusion of S5a's UIM domains to a ubiquitination reaction containing 26S proteasomes, UbcH5, an E3 (MuRF1 or CHIP), and a protein substrate, dramatically stimulated its degradation, provided S5a was present during ubiquitination. Mass spectrometry showed that S5a and GST–UIM prevented the formation of Ub forks without affecting synthesis of standard isopeptide linkages. The forked Ub chains bind poorly to 26S proteasomes unlike those synthesized with S5a present or linked to Lys63 or Lys48 chains. Thus, S5a (and presumably certain other UIM proteins) function with certain E3/E2 pairs to ensure synthesis of efficiently degraded non‐forked Ub conjugates.     

Molecular Basis for Impaired DNA Damage Response Function Associated with the RAP80 ΔE81 Defect

Signal transduction within the DNA damage response is driven by the flux of protein-protein interaction cascades that ultimately recruit repair complexes to sites of damage. The protein RAP80 plays a central role in the damage response by targeting BRCA1/BRCA2 tumor suppressors to DNA damage foci through multivalent binding of Lys-63-linked polyubiquitin chains. Mutations within the high penetrance BRCA1/BRCA2 genes account for ∼20% of familial breast cancers. The genetic basis for the remaining cancers remains unknown, but may involve defects in binding partners for BRCA1 and BRCA2 that lead to impaired targeting to foci and a concomitant role in the pathogenesis of cancer. Recently, an in-frame deletion mutation (ΔE81) in a conserved region from the first ubiquitin interaction motif of RAP80 has been linked to an increase in chromosomal abnormalities. Using NMR spectroscopy, we demonstrate that the N-cap motif within the α-helix of the first ubiquitin interaction motif from ΔE81 undergoes a structural frameshift that leads to abolishment of multivalent binding of polyubiquitin chains. Loss of this single glutamate residue disrupts favorable electrostatic interactions between RAP80 and ubiquitin, establishing a plausible molecular basis for a potential predisposition to cancer unrelated to mutations within BRCA1/BRCA2 genes.     

CtIP protein dimerization is critical for its recruitment to chromosomal DNA double-stranded breaks

CtIP (CtBP-interacting protein) associates with BRCA1 and the Mre11-Rad50-Nbs1 (MRN) complex and plays an essential role in homologous recombination (HR)-mediated DNA double-stranded break (DSB) repair. It has been described that CtIP forms dimers in mammalian cells, but the biological significance is not clear. In this study, we identified a conserved motif in the N terminus of CtIP, which is required for dimer formation. We further showed that CtIP mutants impaired in forming dimers are strongly defective in HR, end resection, and activation of the ataxia telangiectasia and Rad3-related pathway, without notable change of CtIP interactions with BRCA1 or Nbs1. In addition to HR, CtIP dimerization is also required for microhomology-mediated end joining. Live cell imaging of enhanced GFP-tagged CtIP demonstrates that the CtIP dimerization mutant fails to be localized to DSBs, whereas placing a heterologous dimerization motif to the dimerization mutant restores CtIP recruitment to DSBs. These studies suggest that CtIP dimer formation is essential for its recruitment to DSBs on chromatin upon DNA damage. Furthermore, DNA damage-induced phosphorylation of CtIP is significantly reduced in the CtIP dimerization mutants. Therefore, in addition to the C-terminal conserved domains critical for CtIP function, the dimerization motif on the N terminus of CtIP is also conserved and essential for its function in DNA damage responses. The severe repair defects of CtIP dimerization mutants are likely due to the failure in localization to chromosomal DSBs upon DNA damage.     

Regulatory ubiquitylation in response to DNA double-strand breaks

DNA double-strand breaks (DSBs) are highly cytolethal DNA lesions. In response to DSBs, cells initiate a complex response that minimizes their deleterious impact on cellular and organismal physiology. In this review, we discuss the discovery of a regulatory ubiquitylation system that modifies the chromatin that surrounds DNA lesions. This pathway is under the control of RNF8 and RNF168, two E3 ubiquitin ligases that cooperate with UBC13 to promote the relocalization of 53BP1 and BRCA1 to sites of DNA damage. RNF8 and RNF168 orchestrate the recruitment of DNA damage response proteins by catalyzing the ubiquitylation of H2A-type histones and the formation of K63-linked ubiquitin chains on damaged chromatin. Finally, we identify some unresolved issues raised by the discovery of this pathway and discuss the implications of DNA damage-induced ubiquitylation in human disease and development.     

Recruitment of proteins to DNA double-strand breaks: MDC1 directly recruits RAP80

DNA double-strand breaks (DSBs) are the most severe type of DNA damage. Occurrence of DSBs in the cell activates the DNA damage response (DDR), which involves signaling cascades that sense and respond to the damage. Promptly after DSB induction, DDR proteins accumulate surrounding both DNA ends and form microscopically-visible foci. Recently, we demonstrated that the key DDR protein MDC1 directly binds RAP80, an additional DDR protein that recruits BRCA1 to DSBs. We provided evidences that the MDC1-RAP80 interaction depends on a ubiquitylation event on K-1977 of MDC1. However, it remained unknown whether K-1977 of MDC1 is required for the recruitment of RAP80 to DSBs. Here we show that K-1977 of MDC1 is necessary for focus formation by RAP80. Nevertheless, it has not effect on focus formation by γ-H2AX, MDC1 or 53BP1. The results imply a role for the MDC1-RAP80 interaction in focus formation by the RAP80-BRCA1 complex. In light of these recent results we discuss several aspects of the complexity of focus formation and present a model for the involvement of individual and complex recruitment mechanisms in focus formation.     

SUMO pathway dependent recruitment of cellular repressors to herpes simplex virus type 1 genomes

Components of promyelocytic leukaemia (PML) nuclear bodies (ND10) are recruited to sites associated with herpes simplex virus type 1 (HSV-1) genomes soon after they enter the nucleus. This cellular response is linked to intrinsic antiviral resistance and is counteracted by viral regulatory protein ICP0. We report that the SUMO interaction motifs of PML, Sp100 and hDaxx are required for recruitment of these repressive proteins to HSV-1 induced foci, which also contain SUMO conjugates and PIAS2β, a SUMO E3 ligase. SUMO modification of PML and elements of its tripartite motif (TRIM) are also required for recruitment in cells lacking endogenous PML. Mutants of PML isoform I and hDaxx that are not recruited to virus induced foci are unable to reproduce the repression of ICP0 null mutant HSV-1 infection mediated by their wild type counterparts. We conclude that recruitment of ND10 components to sites associated with HSV-1 genomes reflects a cellular defence against invading pathogen DNA that is regulated through the SUMO modification pathway.     

S. cerevisiae Mre11 recruits conjugated SUMO moieties to facilitate the assembly and function of the Mre11-Rad50-Xrs2 complex

Double-strand breaks (DSBs) in chromosomes are the most challenging type of DNA damage. The yeast and mammalian Mre11-Rad50-Xrs2/Nbs1 (MRX/N)-Sae2/Ctp1 complex catalyzes the resection of DSBs induced by secondary structures, chemical adducts or covalently-attached proteins. MRX/N also initiates two parallel DNA damage responses—checkpoint phosphorylation and global SUMOylation—to boost a cell''s ability to repair DSBs. However, the molecular mechanism of this SUMO-mediated response is not completely known. In this study, we report that Saccharomyces cerevisiae Mre11 can non-covalently recruit the conjugated SUMO moieties, particularly the poly-SUMO chain. Mre11 has two evolutionarily-conserved SUMO-interacting motifs, Mre11^SIM1 and Mre11^SIM2, which reside on the outermost surface of Mre11. Mre11^SIM1 is indispensable for MRX assembly. Mre11^SIM2 non-covalently links MRX with the SUMO enzymes (E2/Ubc9 and E3/Siz2) to promote global SUMOylation of DNA repair proteins. Mre11^SIM2 acts independently of checkpoint phosphorylation. During meiosis, the mre11^SIM2 mutant, as for mre11S, rad50S and sae2Δ, allows initiation but not processing of Spo11-induced DSBs. Using MRX and DSB repair as a model, our work reveals a general principle in which the conjugated SUMO moieties non-covalently facilitate the assembly and functions of multi-subunit protein complexes.     

HP1 promotes tumor suppressor BRCA1 functions during the DNA damage response

The DNA damage response (DDR) involves both the control of DNA damage repair and signaling to cell cycle checkpoints. Therefore, unraveling the underlying mechanisms of the DDR is important for understanding tumor suppression and cellular resistance to clastogenic cancer therapeutics. Because the DDR is likely to be influenced by chromatin regulation at the sites of DNA damage, we investigated the role of heterochromatin protein 1 (HP1) during the DDR process. We monitored double-strand breaks (DSBs) using the γH2AX foci marker and found that depleting cells of HP1 caused genotoxic stress, a delay in the repair of DSBs and elevated levels of apoptosis after irradiation. Furthermore, we found that these defects in repair were associated with impaired BRCA1 function. Depleting HP1 reduced recruitment of BRCA1 to DSBs and caused defects in two BRCA1-mediated DDR events: (i) the homologous recombination repair pathway and (ii) the arrest of cell cycle at the G₂/M checkpoint. In contrast, depleting HP1 from cells did not affect the non-homologous end-joining (NHEJ) pathway: instead it elevated the recruitment of the 53BP1 NHEJ factor to DSBs. Notably, all three subtypes of HP1 seemed to be almost equally important for these DDR functions. We suggest that the dynamic interaction of HP1 with chromatin and other DDR factors could determine DNA repair choice and cell fate after DNA damage. We also suggest that compromising HP1 expression could promote tumorigenesis by impairing the function of the BRCA1 tumor suppressor.     

A PP1-binding motif present in BRCA1 plays a role in its DNA repair function

Protein phosphatase 1alpha (PP1alpha) regulates phosphorylation of BRCA1, which contains a PP1-binding motif (898)KVTF(901). Mutation of this motif greatly reduces the interaction between BRCA1 and PP1alpha. Here we show that mutation of the PP1-binding motif abolishes the ability of BRCA1 to enhance survival of Brca1-deficient mouse mammary tumor cells after DNA damage. The Rad51 focus formation and comet assays revealed that the DNA repair function of BRCA1 was impaired when the PP1-binding motif was mutated. Analysis of subnuclear localization of GFP-tagged BRCA1 demonstrated that mutation of the PP1-binding motif affected BRCA1 redistribution in response to DNA damage. BRCA1 is required for the formation of Rad51 subnuclear foci after DNA damage. Mutation of the PP1-binding motif in BRCA1 also affected recruitment of Rad51 to sites of DNA damage. Consistent with these findings, knockdown of PP1alpha in BRCA1-proficient cells by small interfering RNA also significantly reduced Rad51 focus formation induced by DNA damage. Further analysis indicated that mutation of the PP1-binding motif compromised BRCA1 activities in homologous recombination. Altogether, our data implicate that interaction with PP1alpha is important for BRCA1 function in DNA repair.     

The de-ubiquitylating enzymes USP26 and USP37 regulate homologous recombination by counteracting RAP80

The faithful repair of DNA double-strand breaks (DSBs) is essential to safeguard genome stability. DSBs elicit a signaling cascade involving the E3 ubiquitin ligases RNF8/RNF168 and the ubiquitin-dependent assembly of the BRCA1-Abraxas-RAP80-MERIT40 complex. The association of BRCA1 with ubiquitin conjugates through RAP80 is known to be inhibitory to DSB repair by homologous recombination (HR). However, the precise regulation of this mechanism remains poorly understood. Through genetic screens we identified USP26 and USP37 as key de-ubiquitylating enzymes (DUBs) that limit the repressive impact of RNF8/RNF168 on HR. Both DUBs are recruited to DSBs where they actively remove RNF168-induced ubiquitin conjugates. Depletion of USP26 or USP37 disrupts the execution of HR and this effect is alleviated by the simultaneous depletion of RAP80. We demonstrate that USP26 and USP37 prevent excessive spreading of RAP80-BRCA1 from DSBs. On the other hand, we also found that USP26 and USP37 promote the efficient association of BRCA1 with PALB2. This suggests that these DUBs limit the ubiquitin-dependent sequestration of BRCA1 via the BRCA1-Abraxas-RAP80-MERIT40 complex, while promoting complex formation and cooperation of BRCA1 with PALB2-BRCA2-RAD51 during HR. These findings reveal a novel ubiquitin-dependent mechanism that regulates distinct BRCA1-containing complexes for efficient repair of DSBs by HR.     

Two redundant ubiquitin‐dependent pathways of BRCA1 localization to DNA damage sites

The tumor suppressor BRCA1 accumulates at sites of DNA damage in a ubiquitin‐dependent manner. In this work, we revisit the role of RAP80 in promoting BRCA1 recruitment to damaged chromatin. We find that RAP80 acts redundantly with the BRCA1 RING domain to promote BRCA1 recruitment to DNA damage sites. We show that that RNF8 E3 ligase acts upstream of both the RAP80‐ and RING‐dependent activities, whereas RNF168 acts uniquely upstream of the RING domain. BRCA1 RING mutations that do not impact BARD1 interaction, such as the E2 binding‐deficient I26A mutation, render BRCA1 unable to accumulate at DNA damage sites in the absence of RAP80. Cells that combine BRCA1 I26A and mutations that disable the RAP80–BRCA1 interaction are hypersensitive to PARP inhibition and are unable to form RAD51 foci. Our results suggest that in the absence of RAP80, the BRCA1 E3 ligase activity is necessary for recognition of histone H2A Lys13/Lys15 ubiquitylation by BARD1, although we cannot rule out the possibility that the BRCA1 RING facilitates ubiquitylated nucleosome recognition in other ways.     

Ubiquitin-activating enzyme UBA1 is required for cellular response to DNA damage

The cellular DNA damage response (DDR) machinery that maintains genomic integrity and prevents severe pathologies, including cancer, is orchestrated by signaling through protein modifications. Protein ubiquitylation regulates repair of DNA double-strand breaks (DSBs), toxic lesions caused by various metabolic as well as environmental insults such as ionizing radiation (IR). Whereas several components of the DSB-evoked ubiquitylation cascade have been identified, including RNF168 and BRCA1 ubiquitin ligases, whose genetic defects predispose to a syndrome mimicking ataxia-telangiectasia and cancer, respectively, the identity of the apical E1 enzyme involved in DDR has not been established. Here, we identify ubiquitin-activating enzyme UBA1 as the E1 enzyme required for responses to IR and replication stress in human cells. We show that siRNA-mediated knockdown of UBA1, but not of another UBA family member UBA6, impaired formation of both ubiquitin conjugates at the sites of DNA damage and IR-induced foci (IRIF) by the downstream components of the DSB response pathway, 53BP1 and BRCA1. Furthermore, chemical inhibition of UBA1 prevented IRIF formation and severely impaired DSB repair and formation of 53BP1 bodies in G₁, a marker of response to replication stress. In contrast, the upstream steps of DSB response, such as phosphorylation of histone H2AX and recruitment of MDC1, remained unaffected by UBA1 depletion. Overall, our data establish UBA1 as the apical enzyme critical for ubiquitylation-dependent signaling of both DSBs and replication stress in human cells, with implications for maintenance of genomic integrity, disease pathogenesis and cancer treatment.     

The structures of ubiquitin conjugates of yeast Iso-2-cytochrome c.

Ubiquitin (Ub) conjugates to Saccharomyces cerevisiae iso-2-cytochrome c were formed in vitro in a rabbit reticulocyte extract (Fraction II). In the presence of ubiquitin-aldehyde, used to inhibit ubiquitin-protein isopeptidases in Fraction II, mono-, di-, and triubiquitinated cytochrome c conjugates accumulated in a 1.2:1.0:0.2 molar ratio. CNBr digestions showed that, in all three conjugates, Ub attachment was within the first 73 amino acids of the cytochrome c. For the two most abundant conjugates, this region was further narrowed to the first 30 residues by peptide mapping with Staphylococcus aureus V8 protease. N-terminal protein sequencing identified Lys-13 as the major ubiquitination site in each conjugate. For di- and triubiquitinated iso-2-cytochrome c, this suggested that Ub2 and Ub3 multiubiquitin chains extend from Lys-13. This conclusion was supported by a variation of protein sequencing in which polypeptides recovered after Edman degradation were analyzed to determine at which cycle(s) radiolabeled Ub or Ubn was cleaved from the conjugate. Because of the sensitivity afforded by the use of 125I-Ub in this "stutter-step" sequencing method, minor ubiquitination at Lys-8 also was detected. Thus, Ub2-iso-2-cytochrome c conjugates contain mostly Ub2 at Lys-13 with a small fraction of conjugates having single Ubs on 2 residues, Lys-8 and Lys-13. Similarly, Ub3-iso-2-cytochrome c predominantly has a Ub3 chain on Lys-13, although minor species with combinations of Ub1 and Ub2 distributed on Lys-8 and Lys-13 also may be present. This specificity is discussed in the context of iso-2-cytochrome c structure.