ABSTRACT
Transcription-blocking DNA lesions are specifically targeted by transcription-coupled nucleotide excision repair (TC-NER), which removes a broad spectrum of DNA lesions to preserve transcriptional output and thereby cellular homeostasis to counteract aging. TC-NER is initiated by the stalling of RNA polymerase II at DNA lesions, which triggers the assembly of the TC-NER-specific proteins CSA, CSB and UVSSA. CSA, a WD40-repeat containing protein, is the substrate receptor subunit of a cullin-RING ubiquitin ligase complex composed of DDB1, CUL4A/B and RBX1 (CRL4CSA). Although ubiquitination of several TC-NER proteins by CRL4CSA has been reported, it is still unknown how this complex is regulated. To unravel the dynamic molecular interactions and the regulation of this complex, we apply a single-step protein-complex isolation coupled to mass spectrometry analysis and identified DDA1 as a CSA interacting protein. Cryo-EM analysis shows that DDA1 is an integral component of the CRL4CSA complex. Functional analysis reveals that DDA1 coordinates ubiquitination dynamics during TC-NER and is required for efficient turnover and progression of this process.
Subject(s)
DNA Repair , DNA-Binding Proteins , Transcription, Genetic , Ubiquitination , Humans , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Cullin Proteins/metabolism , Cullin Proteins/genetics , DNA Repair Enzymes/metabolism , DNA Repair Enzymes/genetics , Cryoelectron Microscopy , HEK293 Cells , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Poly-ADP-Ribose Binding Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/genetics , DNA Damage , RNA Polymerase II/metabolism , Protein Binding , Excision Repair , Carrier Proteins , DNA Helicases , Transcription Factors , Receptors, Interleukin-17ABSTRACT
UvrD is a helicase vital for DNA replication and quality control processes. In its monomeric state, UvrD exhibits limited helicase activity, necessitating either dimerization or assistance from an accessory protein to efficiently unwind DNA. Within the DNA mismatch repair pathway, MutL plays a pivotal role in relaying the repair signal, enabling UvrD to unwind DNA from the strand incision site up to and beyond the mismatch. Although this interdependence is well-established, the precise mechanism of activation and the specific MutL-UvrD interactions that trigger helicase activity remain elusive. To address these questions, we employed site-specific crosslinking techniques using single-cysteine variants of MutL and UvrD followed by functional assays. Our investigation unveils that the C-terminal domain of MutL not only engages with UvrD but also acts as a self-sufficient activator of UvrD helicase activity on DNA substrates with 3'-single-stranded tails. Especially when MutL is covalently attached to the 2B or 1B domain the tail length can be reduced to a minimal substrate of 5 nucleotides without affecting unwinding efficiency.
Subject(s)
DNA Helicases , MutL Proteins , DNA/chemistry , DNA Helicases/chemistry , DNA Helicases/genetics , MutL Proteins/chemistry , MutL Proteins/genetics , Protein Binding , Protein Domains , Mesylates/chemistry , Cross-Linking Reagents/chemistryABSTRACT
The ubiquitin-specific protease (USP) family of deubiquitinases (DUBs) controls cellular ubiquitin-dependent signaling events. This generates therapeutic potential, with active-site inhibitors in preclinical and clinical studies. Understanding of the USP active site is primarily guided by USP7 data, where the catalytic triad consists of cysteine, histidine, and a third residue (third critical residue), which polarizes the histidine through a hydrogen bond. A conserved aspartate (fourth critical residue) is directly adjacent to this third critical residue. Although both critical residues accommodate catalysis in USP2, these residues have not been comprehensively investigated in other USPs. Here, we quantitatively investigate their roles in five USPs. Although USP7 relies on the third critical residue for catalysis, this residue is dispensable in USP1, USP15, USP40, and USP48, where the fourth critical residue is vital instead. Furthermore, these residues vary in importance for nucleophilic attack. The diverging catalytic mechanisms of USP1 and USP7 are independent of substrate and retained in cells for USP1. This unexpected variety of catalytic mechanisms in this well-conserved protein family may generate opportunities for selective targeting of individual USPs.
Subject(s)
Histidine , Ubiquitin-Specific Proteases , Ubiquitin-Specific Proteases/genetics , Ubiquitin-Specific Proteases/metabolism , Ubiquitin-Specific Peptidase 7/genetics , Ubiquitin-Specific Peptidase 7/metabolism , CatalysisABSTRACT
The DNA mismatch repair protein MutSα recognizes wrongly incorporated DNA bases and initiates their correction during DNA replication. Dysfunctions in mismatch repair lead to a predisposition to cancer. Here, we study the homozygous mutation V63E in MSH2 that was found in the germline of a patient with suspected constitutional mismatch repair deficiency syndrome who developed colorectal cancer before the age of 30. Characterization of the mutant in mouse models, as well as slippage and repair assays, shows a mildly pathogenic phenotype. Using cryogenic electron microscopy and surface plasmon resonance, we explored the mechanistic effect of this mutation on MutSα function. We discovered that V63E disrupts a previously unappreciated interface between the mismatch binding domains (MBDs) of MSH2 and MSH6 and leads to reduced DNA binding. Our research identifies this interface as a 'safety lock' that ensures high-affinity DNA binding to increase replication fidelity. Our mechanistic model explains the hypomorphic phenotype of the V63E patient mutation and other variants in the MBD interface.
Subject(s)
DNA Mismatch Repair , DNA Repair , MutS Homolog 2 Protein , Animals , Mice , DNA/chemistry , Mutation , MutS Homolog 2 Protein/metabolismABSTRACT
Nucleosome assembly requires the coordinated deposition of histone complexes H3-H4 and H2A-H2B to form a histone octamer on DNA. In the current paradigm, specific histone chaperones guide the deposition of first H3-H4 and then H2A-H2B. Here, we show that the acidic domain of DNA repair factor APLF (APLFAD) can assemble the histone octamer in a single step and deposit it on DNA to form nucleosomes. The crystal structure of the APLFAD-histone octamer complex shows that APLFAD tethers the histones in their nucleosomal conformation. Mutations of key aromatic anchor residues in APLFAD affect chaperone activity in vitro and in cells. Together, we propose that chaperoning of the histone octamer is a mechanism for histone chaperone function at sites where chromatin is temporarily disrupted.
Subject(s)
Histones , Nucleosomes , DNA/chemistry , DNA Repair , Histone Chaperones/genetics , Histone Chaperones/metabolism , Histones/metabolism , Molecular Chaperones/geneticsABSTRACT
Ubiquitin specific protease USP15 is a deubiquitinating enzyme reported to regulate several biological and cellular processes, including TGF-ß signaling, regulation of immune response, neuro-inflammation and mRNA splicing. Here we study the USP15 D1D2 catalytic domain and present the crystal structure in its catalytically-competent conformation. We compare this apo-structure to a previous misaligned state in the same crystal lattice. In both structures, mitoxantrone, an FDA approved antineoplastic drug and a weak inhibitor of USP15 is bound, indicating that it is not responsible for inducing a switch in the conformation of active site cysteine in the USP15 D1D2 structure. Instead, mitoxantrone contributes to crystal packing, by forming a stack of 12 mitoxantrone molecules. We believe this reflects how mitoxantrone can be responsible for e.g. nuclear condensate partitioning. We conclude that USP15 can switch between active and inactive states in the absence of ubiquitin, and that this is independent of mitoxantrone binding. These insights can be important for future drug discovery targeting USP15.
Subject(s)
Mitoxantrone , Ubiquitin-Specific Proteases , Catalytic Domain , Protein Binding , Ubiquitin/metabolism , Ubiquitin-Specific Proteases/chemistry , Ubiquitin-Specific Proteases/genetics , Ubiquitin-Specific Proteases/metabolismABSTRACT
AlphaFold is the most ground-breaking application of AI in science so far; it will revolutionize structural biology, but caution is warranted.
Subject(s)
Artificial Intelligence , BiologyABSTRACT
BRCA1-associated protein 1 (BAP1) is a tumor suppressor and its loss can result in mesothelioma, uveal and cutaneous melanoma, clear cell renal cell carcinoma and bladder cancer. BAP1 is a deubiquitinating enzyme of the UCH class that has been implicated in various cellular processes like cell growth, cell cycle progression, ferroptosis, DNA damage response and ER metabolic stress response. ASXL proteins activate BAP1 by forming the polycomb repressive deubiquitinase (PR-DUB) complex which acts on H2AK119ub1. Besides the ASXL proteins, BAP1 is known to interact with an established set of additional proteins. Here, we identify novel BAP1 interacting proteins in the cytoplasm by expressing GFP-tagged BAP1 in an endogenous BAP1 deficient cell line using affinity purification followed by mass spectrometry (AP-MS) analysis. Among these novel interacting proteins are Histone acetyltransferase 1 (HAT1) and all subunits of the heptameric coat protein complex I (COPI) that is involved in vesicle formation and protein cargo binding and sorting. We validate that the HAT1 and COPI interactions occur at endogenous levels but find that this interaction with COPI is not mediated through the C-terminal KxKxx cargo sorting signals of the COPI complex.
Subject(s)
Coat Protein Complex I/metabolism , Cytoplasm/metabolism , Histone Acetyltransferases/metabolism , Neoplasms/metabolism , Proteomics/methods , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Ubiquitin Thiolesterase/genetics , Ubiquitin Thiolesterase/metabolism , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Gene Knockout Techniques , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Mass Spectrometry , Mutation , Recombinant Proteins/metabolismABSTRACT
DNA mismatch repair detects and removes mismatches from DNA by a conserved mechanism, reducing the error rate of DNA replication by 100- to 1,000-fold. In this process, MutS homologs scan DNA, recognize mismatches and initiate repair. How the MutS homologs selectively license repair of a mismatch among millions of matched base pairs is not understood. Here we present four cryo-EM structures of Escherichia coli MutS that provide snapshots, from scanning homoduplex DNA to mismatch binding and MutL activation via an intermediate state. During scanning, the homoduplex DNA forms a steric block that prevents MutS from transitioning into the MutL-bound clamp state, which can only be overcome through kinking of the DNA at a mismatch. Structural asymmetry in all four structures indicates a division of labor between the two MutS monomers. Together, these structures reveal how a small conformational change from the homoduplex- to heteroduplex-bound MutS acts as a licensing step that triggers a dramatic conformational change that enables MutL binding and initiation of the repair cascade.
Subject(s)
DNA/ultrastructure , Escherichia coli Proteins/ultrastructure , MutL Proteins/ultrastructure , MutS DNA Mismatch-Binding Protein/ultrastructure , Protein Conformation , Cryoelectron Microscopy , DNA/genetics , DNA Mismatch Repair/genetics , DNA Repair/genetics , DNA Replication/genetics , Escherichia coli/genetics , Escherichia coli/ultrastructure , Escherichia coli Proteins/genetics , MutL Proteins/genetics , MutS DNA Mismatch-Binding Protein/geneticsABSTRACT
During DNA replication, the deubiquitinating enzyme USP1 limits the recruitment of translesion polymerases by removing ubiquitin marks from PCNA to allow specific regulation of the translesion synthesis (TLS) pathway. USP1 activity depends on an allosteric activator, UAF1, and this is tightly controlled. In comparison to paralogs USP12 and USP46, USP1 contains three defined inserts and lacks the second WDR20-mediated activation step. Here we show how inserts L1 and L3 together limit intrinsic USP1 activity and how this is relieved by UAF1. Intriguingly, insert L1 also conveys substrate-dependent increase in USP1 activity through DNA and PCNA interactions, in a process that is independent of UAF1-mediated activation. This study establishes insert L1 as an important regulatory hub within USP1 necessary for both substrate-mediated activity enhancement and allosteric activation upon UAF1 binding.
Subject(s)
Nuclear Proteins , Ubiquitin-Specific Proteases , Allosteric Regulation , DNA Repair , Nuclear Proteins/metabolism , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , Ubiquitin , Ubiquitin-Specific Proteases/genetics , Ubiquitin-Specific Proteases/metabolism , UbiquitinationABSTRACT
Functional analysis of lysine 27-linked ubiquitin chains (K27Ub) is difficult due to the inability to make them through enzymatic methods and due to a lack of model tools and substrates. Here we generate a series of ubiquitin (Ub) tools to study how the deubiquitinase UCHL3 responds to K27Ub chains in comparison to lysine 63-linked chains and mono-Ub. From a crystal structure of a complex between UCHL3 and synthetic K27Ub2, we unexpectedly discover that free K27Ub2 and K27Ub2-conjugated substrates are natural inhibitors of UCHL3. Using our Ub tools to profile UCHL3's activity, we generate a quantitative kinetic model of the inhibitory mechanism and we find that K27Ub2 can inhibit UCHL3 covalently, by binding to its catalytic cysteine, and allosterically, by locking its catalytic loop tightly in place. Based on this inhibition mechanism, we propose that UCHL3 and K27Ub chains likely sense and regulate each other in cells.
Subject(s)
Ubiquitin Thiolesterase/metabolism , Ubiquitins/metabolism , Allosteric Regulation , Crystallography, X-Ray , Humans , Kinetics , Models, Molecular , Protein Conformation , Substrate Specificity , Ubiquitin Thiolesterase/antagonists & inhibitors , Ubiquitin Thiolesterase/chemistry , Ubiquitination , Ubiquitins/chemistryABSTRACT
Hepatic abundance of the low-density lipoprotein receptor (LDLR) is a critical determinant of circulating plasma LDL cholesterol levels and hence development of coronary artery disease. The sterol-responsive E3 ubiquitin ligase inducible degrader of the LDLR (IDOL) specifically promotes ubiquitination and subsequent lysosomal degradation of the LDLR and thus controls cellular LDL uptake. IDOL contains an extended N-terminal FERM (4.1 protein, ezrin, radixin, and moesin) domain, responsible for substrate recognition and plasma membrane association, and a second C-terminal RING domain, responsible for the E3 ligase activity and homodimerization. As IDOL is a putative lipid-lowering drug target, we investigated the molecular details of its substrate recognition. We produced and isolated full-length IDOL protein, which displayed high autoubiquitination activity. However, in vitro ubiquitination of its substrate, the intracellular tail of the LDLR, was low. To investigate the structural basis for this, we determined crystal structures of the extended FERM domain of IDOL and multiple conformations of its F3ab subdomain. These reveal the archetypal F1-F2-F3 trilobed FERM domain structure but show that the F3c subdomain orientation obscures the target-binding site. To substantiate this finding, we analyzed the full-length FERM domain and a series of truncated FERM constructs by small-angle X-ray scattering (SAXS). The scattering data support a compact and globular core FERM domain with a more flexible and extended C-terminal region. This flexibility may explain the low activity in vitro and suggests that IDOL may require activation for recognition of the LDLR.
Subject(s)
Receptors, LDL/metabolism , Ubiquitin-Protein Ligases/metabolism , Binding Sites , FERM Domains , Humans , Models, Molecular , Receptors, LDL/chemistry , Substrate Specificity , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/geneticsABSTRACT
DNA mismatch repair (MMR) maintains genome stability through repair of DNA replication errors. In Escherichia coli, initiation of MMR involves recognition of the mismatch by MutS, recruitment of MutL, activation of endonuclease MutH and DNA strand incision at a hemimethylated GATC site. Here, we studied the mechanism of communication that couples mismatch recognition to daughter strand incision. We investigated the effect of catalytically-deficient Cas9 as well as stalled RNA polymerase as roadblocks placed on DNA in between the mismatch and GATC site in ensemble and single molecule nanomanipulation incision assays. The MMR proteins were observed to incise GATC sites beyond a roadblock, albeit with reduced efficiency. This residual incision is completely abolished upon shortening the disordered linker regions of MutL. These results indicate that roadblock bypass can be fully attributed to the long, disordered linker regions in MutL and establish that communication during MMR initiation occurs along the DNA backbone.
Subject(s)
DNA Mismatch Repair/genetics , DNA, Bacterial/genetics , Deoxyribonucleases, Type II Site-Specific/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/genetics , MutL Proteins/metabolism , Base Pair Mismatch/genetics , CRISPR-Associated Protein 9/genetics , DNA Repair Enzymes/metabolism , DNA-Binding Proteins/metabolism , DNA-Directed RNA Polymerases/genetics , Endodeoxyribonucleases/metabolism , Genomic Instability/genetics , MutS DNA Mismatch-Binding Protein/metabolismABSTRACT
DNA mismatch repair (MMR) corrects mismatches, small insertions and deletions in DNA during DNA replication. While scanning for mismatches, dimers of MutS embrace the DNA helix with their lever and clamp domains. Previous studies indicated generic flexibility of the lever and clamp domains of MutS prior to DNA binding, but whether this was important for MutS function was unknown. Here, we present a novel crystal structure of DNA-free Escherichia coli MutS. In this apo-structure, the clamp domains are repositioned due to kinking at specific sites in the coiled-coil region in the lever domains, suggesting a defined hinge point. We made mutations at the coiled-coil hinge point. The mutants made to disrupt the helical fold at the kink site diminish DNA binding, whereas those made to increase stability of coiled-coil result in stronger DNA binding. These data suggest that the site-specific kinking of the coiled-coil in the lever domain is important for loading of this ABC-ATPase on DNA.
Subject(s)
Apoproteins/chemistry , DNA, Bacterial/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/genetics , MutS DNA Mismatch-Binding Protein/chemistry , Amino Acid Sequence , Apoproteins/genetics , Apoproteins/metabolism , Binding Sites , Cloning, Molecular , Crystallography, X-Ray , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Kinetics , Models, Molecular , MutS DNA Mismatch-Binding Protein/genetics , MutS DNA Mismatch-Binding Protein/metabolism , Mutagenesis, Site-Directed , Mutation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Structure-Activity RelationshipABSTRACT
Ubiquitination of chromatin by modification of histone H2A is a critical step in both regulation of DNA repair and regulation of cell fate. These very different outcomes depend on the selective modification of distinct lysine residues in H2A, each by a specific E3 ligase. While polycomb PRC1 complexes modify K119, resulting in gene silencing, the E3 ligase RNF168 modifies K13/15, which is a key event in the response to DNA double-strand breaks. The molecular origin of ubiquitination site specificity by these related E3 enzymes is one of the open questions in the field. Using a combination of NMR spectroscopy, crosslinking mass-spectrometry, mutagenesis and data-driven modelling, here we show that RNF168 binds the acidic patch on the nucleosome surface, directing the E2 to the target lysine. The structural model highlights the role of E3 and nucleosome in promoting ubiquitination and provides a basis for understanding and engineering of chromatin ubiquitination specificity.
Subject(s)
Histones/chemistry , Ubiquitin-Protein Ligases/chemistry , Cell Differentiation , DNA Repair , Histones/metabolism , Humans , Models, Molecular , Protein Domains , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/physiology , UbiquitinationABSTRACT
Ubiquitin-specific proteases (USPs) are an important class of deubiquitinating enzymes (DUBs) that carry out critical roles in cellular physiology and are regulated at multiple levels. Quantitative characterization of USP activity is crucial for mechanistic understanding of USP function and regulation. This requires kinetic analysis using in vitro activity assays on minimal and natural substrates with purified proteins. In this chapter we give advice for efficient design of USP constructs and their optimal expression, followed by a series of purification strategies. We then present protocols for studying USP activity quantitatively on minimal and more natural substrates, and we discuss how to include possible regulatory elements such as internal USP domains or external interacting proteins. Lastly, we examine different binding assays for studying USP interactions and discuss how these can be included in full kinetic analyses.
Subject(s)
Ubiquitin-Specific Proteases/metabolism , Animals , Enzyme Assays/methods , Humans , Kinetics , Protein Interaction Mapping/methods , Protein Interaction Maps , Substrate Specificity , Ubiquitin/metabolism , Ubiquitin-Specific Proteases/isolation & purificationABSTRACT
USP7 is a highly abundant deubiquitinating enzyme (DUB), involved in cellular processes including DNA damage response and apoptosis. USP7 has an unusual catalytic mechanism, where the low intrinsic activity of the catalytic domain (CD) increases when the C-terminal Ubl domains (Ubl45) fold onto the CD, allowing binding of the activating C-terminal tail near the catalytic site. Here we delineate how the target protein promotes the activation of USP7. Using NMR analysis and biochemistry we describe the order of activation steps, showing that ubiquitin binding is an instrumental step in USP7 activation. Using chemically synthesised p53-peptides we also demonstrate how the correct ubiquitinated substrate increases catalytic activity. We then used transient reaction kinetic modelling to define how the USP7 multistep mechanism is driven by target recognition. Our data show how this pleiotropic DUB can gain specificity for its cellular targets.
Subject(s)
Protein Processing, Post-Translational , Ubiquitin-Specific Peptidase 7/metabolism , Ubiquitin/metabolism , Carbon Isotopes/chemistry , Catalytic Domain/genetics , Enzyme Assays/methods , Kinetics , Models, Chemical , Mutagenesis, Site-Directed , Nitrogen Isotopes/chemistry , Nuclear Magnetic Resonance, Biomolecular/methods , Peptides/chemistry , Peptides/metabolism , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Substrate Specificity , Surface Plasmon Resonance , Tumor Suppressor Protein p53/chemistry , Ubiquitin/chemistry , Ubiquitin-Specific Peptidase 7/chemistry , Ubiquitin-Specific Peptidase 7/genetics , Ubiquitin-Specific Peptidase 7/isolation & purificationABSTRACT
A common strategy for exploring the biological roles of deubiquitinating enzymes (DUBs) in different pathways is to study the effects of replacing the wild-type DUB with a catalytically inactive mutant in cells. We report here that a commonly studied DUB mutation, in which the catalytic cysteine is replaced with alanine, can dramatically increase the affinity of some DUBs for ubiquitin. Overexpression of these tight-binding mutants thus has the potential to sequester cellular pools of monoubiquitin and ubiquitin chains. As a result, cells expressing these mutants may display unpredictable dominant negative physiological effects that are not related to loss of DUB activity. The structure of the SAGA DUB module bound to free ubiquitin reveals the structural basis for the 30-fold higher affinity of Ubp8C146A for ubiquitin. We show that an alternative option, substituting the active site cysteine with arginine, can inactivate DUBs while also decreasing the affinity for ubiquitin.
Subject(s)
Deubiquitinating Enzymes/genetics , Endopeptidases/genetics , Saccharomyces cerevisiae Proteins/genetics , Trans-Activators/genetics , Ubiquitin-Specific Proteases/genetics , Alanine/genetics , Amino Acid Substitution/genetics , Carrier Proteins/chemistry , Carrier Proteins/genetics , Catalysis , Cysteine/genetics , Deubiquitinating Enzymes/chemistry , Endopeptidases/chemistry , Humans , Mutation/genetics , Protein Conformation , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Trans-Activators/chemistry , Ubiquitin/chemistry , Ubiquitin/genetics , Ubiquitin-Specific Proteases/chemistry , Ubiquitination/geneticsABSTRACT
Deubiquitylating enzymes (DUBs) enhance the dynamics of the versatile ubiquitin (Ub) code by reversing and regulating cellular ubiquitylation processes at multiple levels. Here we discovered that the uncharacterized human protein ZUFSP (zinc finger with UFM1-specific peptidase domain protein/C6orf113/ZUP1), which has been annotated as a potentially inactive UFM1 protease, and its fission yeast homolog Mug105 define a previously unrecognized class of evolutionarily conserved cysteine protease DUBs. Human ZUFSP selectively interacts with and cleaves long K63-linked poly-Ub chains by means of tandem Ub-binding domains, whereas it displays poor activity toward mono- or di-Ub substrates. In cells, ZUFSP is recruited to and regulates K63-Ub conjugates at genotoxic stress sites, promoting chromosome stability upon replication stress in a manner dependent on its catalytic activity. Our findings establish ZUFSP as a new type of linkage-selective cysteine peptidase DUB with a role in genome maintenance pathways.
Subject(s)
Bone Neoplasms/enzymology , DNA Damage , Deubiquitinating Enzymes/metabolism , Genomic Instability , Osteosarcoma/enzymology , Polyubiquitin/metabolism , Retinal Pigment Epithelium/enzymology , Binding Sites , Bone Neoplasms/genetics , Cell Line, Tumor , Deubiquitinating Enzymes/genetics , HEK293 Cells , Humans , Lysine , Osteosarcoma/genetics , Polyubiquitin/genetics , Protein Binding , Protein Interaction Domains and Motifs , Substrate Specificity , UbiquitinationABSTRACT
BRCA1-BARD1-catalyzed ubiquitination of histone H2A is an important regulator of the DNA damage response, priming chromatin for repair by homologous recombination. However, no specific deubiquitinating enzymes (DUBs) are known to antagonize this function. Here we identify ubiquitin specific protease-48 (USP48) as a H2A DUB, specific for the C-terminal BRCA1 ubiquitination site. Detailed biochemical analysis shows that an auxiliary ubiquitin, an additional ubiquitin that itself does not get cleaved, modulates USP48 activity, which has possible implications for its regulation in vivo. In cells we reveal that USP48 antagonizes BRCA1 E3 ligase function and in BRCA1-proficient cells loss of USP48 results in positioning 53BP1 further from the break site and in extended resection lengths. USP48 repression confers a survival benefit to cells treated with camptothecin and its activity acts to restrain gene conversion and mutagenic single-strand annealing. We propose that USP48 promotes genome stability by antagonizing BRCA1 E3 ligase function.