RESUMO
RNF168 plays a central role in the DNA damage response (DDR) by ubiquitylating histone H2A at K13 and K15. These modifications direct BRCA1-BARD1 and 53BP1 foci formation in chromatin, essential for cell-cycle-dependent DNA double-strand break (DSB) repair pathway selection. The mechanism by which RNF168 catalyzes the targeted accumulation of H2A ubiquitin conjugates to form repair foci around DSBs remains unclear. Here, using cryoelectron microscopy (cryo-EM), nuclear magnetic resonance (NMR) spectroscopy, and functional assays, we provide a molecular description of the reaction cycle and dynamics of RNF168 as it modifies the nucleosome and recognizes its ubiquitylation products. We demonstrate an interaction of a canonical ubiquitin-binding domain within full-length RNF168, which not only engages ubiquitin but also the nucleosome surface, clarifying how such site-specific ubiquitin recognition propels a signal amplification loop. Beyond offering mechanistic insights into a key DDR protein, our study aids in understanding site specificity in both generating and interpreting chromatin ubiquitylation.
Assuntos
Nucleossomos , Ubiquitina-Proteína Ligases , Nucleossomos/genética , Microscopia Crioeletrônica , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Histonas/metabolismo , Cromatina/genética , Reparo do DNA , Ubiquitina/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Dano ao DNARESUMO
53BP1 influences genome stability via two independent mechanisms: (1) regulating DNA double-strand break (DSB) repair and (2) enhancing p53 activity. We discovered a protein, Tudor-interacting repair regulator (TIRR), that associates with the 53BP1 Tudor domain and prevents its recruitment to DSBs. Here, we elucidate how TIRR affects 53BP1 function beyond its recruitment to DSBs and biochemically links the two distinct roles of 53BP1. Loss of TIRR causes an aberrant increase in the gene transactivation function of p53, affecting several p53-mediated cell-fate programs. TIRR inhibits the complex formation between the Tudor domain of 53BP1 and a dimethylated form of p53 (K382me2) that is poised for transcriptional activation of its target genes. TIRR mRNA expression levels negatively correlate with the expression of key p53 target genes in breast and prostate cancers. Further, TIRR loss is selectively not tolerated in p53-proficient tumors. Therefore, we establish that TIRR is an important inhibitor of the 53BP1-p53 complex.
Assuntos
Linhagem da Célula/genética , Proteínas de Ligação a RNA/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Sítios de Ligação , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Linhagem da Célula/fisiologia , DNA/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Histonas/metabolismo , Humanos , Ligação Proteica , Proteínas de Ligação a RNA/fisiologia , Domínio Tudor , Proteína Supressora de Tumor p53/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/fisiologiaRESUMO
The ubiquitin system regulates the DNA damage response (DDR) by modifying histone H2A at Lys15 (H2AK15ub) and triggering downstream signaling events. Here, we find that phosphorylation of ubiquitin at Thr12 (pUbT12) controls the DDR by inhibiting the function of 53BP1, a key factor for DNA double-strand break repair by non-homologous end joining (NHEJ). Detectable as a chromatin modification on H2AK15ub, pUbT12 accumulates in nuclear foci and is increased upon DNA damage. Mutating Thr12 prevents the removal of ubiquitin from H2AK15ub by USP51 deubiquitinating enzyme, leading to a pronounced accumulation of ubiquitinated chromatin. Chromatin modified by pUbT12 is inaccessible to 53BP1 but permissive to the homologous recombination (HR) proteins RNF169, RAD51, and the BRCA1/BARD1 complex. Phosphorylation of ubiquitin at Thr12 in the chromatin context is a new histone mark, H2AK15pUbT12, that regulates the DDR by hampering the activity of 53BP1 at damaged chromosomes.
Assuntos
Dano ao DNA/fisiologia , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Cromatina/metabolismo , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Recombinação Homóloga/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Transdução de Sinais/genética , Treonina/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/fisiologia , Ubiquitina/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
The BRCA1-BARD1 tumour suppressor is an E3 ubiquitin ligase necessary for the repair of DNA double-strand breaks by homologous recombination1-10. The BRCA1-BARD1 complex localizes to damaged chromatin after DNA replication and catalyses the ubiquitylation of histone H2A and other cellular targets11-14. The molecular bases for the recruitment to double-strand breaks and target recognition of BRCA1-BARD1 remain unknown. Here we use cryo-electron microscopy to show that the ankyrin repeat and tandem BRCT domains in BARD1 adopt a compact fold and bind to nucleosomal histones, DNA and monoubiquitin attached to H2A amino-terminal K13 or K15, two signals known to be specific for double-strand breaks15,16. We further show that RING domains17 in BRCA1-BARD1 orient an E2 ubiquitin-conjugating enzyme atop the nucleosome in a dynamic conformation, primed for ubiquitin transfer to the flexible carboxy-terminal tails of H2A and variant H2AX. Our work reveals a regulatory crosstalk in which recognition of monoubiquitin by BRCA1-BARD1 at the N terminus of H2A blocks the formation of polyubiquitin chains and cooperatively promotes ubiquitylation at the C terminus of H2A. These findings elucidate the mechanisms of BRCA1-BARD1 chromatin recruitment and ubiquitylation specificity, highlight key functions of BARD1 in both processes and explain how BRCA1-BARD1 promotes homologous recombination by opposing the DNA repair protein 53BP1 in post-replicative chromatin18-22. These data provide a structural framework to evaluate BARD1 variants and help to identify mutations that drive the development of cancer.
Assuntos
Proteína BRCA1/metabolismo , Nucleossomos/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Microscopia Crioeletrônica , Reparo do DNA , Histonas/química , Histonas/metabolismo , Recombinação Homóloga , Humanos , Modelos Moleculares , Mutação , Neoplasias/genética , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/ultraestrutura , Domínios Proteicos , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/ultraestrutura , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/ultraestrutura , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/ultraestruturaRESUMO
In vertebrates, DNA methyltransferase 1 (DNMT1) contributes to preserving DNA methylation patterns, ensuring the stability and heritability of epigenetic marks important for gene expression regulation and the maintenance of cellular identity. Previous structural studies have elucidated the catalytic mechanism of DNMT1 and its specific recognition of hemimethylated DNA. Here, using solution nuclear magnetic resonance spectroscopy and small-angle X-ray scattering, we demonstrate that the N-terminal region of human DNMT1, while flexible, encompasses a conserved globular domain with a novel α-helical bundle-like fold. This work expands our understanding of the structure and dynamics of DNMT1 and provides a structural framework for future functional studies in relation with this new domain.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1 , Animais , Humanos , Domínio Catalítico , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/química , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA , Estrutura Terciária de Proteína , Conformação Proteica em alfa-HéliceRESUMO
The protein 53BP1 plays a central regulatory role in DNA double-strand break repair. 53BP1 relocates to chromatin by recognizing RNF168-mediated mono-ubiquitylation of histone H2A Lys15 in the nucleosome core particle dimethylated at histone H4 Lys20 (NCP-ubme). 53BP1 relocation is terminated by ubiquitin ligases RNF169 and RAD18 via unknown mechanisms. Using nuclear magnetic resonance (NMR) spectroscopy and biochemistry, we show that RNF169 bridges ubiquitin and histone surfaces, stabilizing a pre-existing ubiquitin orientation in NCP-ubme to form a high-affinity complex. This conformational selection mechanism contrasts with the low-affinity binding mode of 53BP1, and it ensures 53BP1 displacement by RNF169 from NCP-ubme. We also show that RAD18 binds tightly to NCP-ubme through a ubiquitin-binding domain that contacts ubiquitin and nucleosome surfaces accessed by 53BP1. Our work uncovers diverse ubiquitin recognition mechanisms in the nucleosome, explaining how RNF168, RNF169, and RAD18 regulate 53BP1 chromatin recruitment and how specificity can be achieved in the recognition of a ubiquitin-modified substrate.
Assuntos
Cromatina/enzimologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Nucleossomos/enzimologia , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Sítios de Ligação , Cromatina/genética , Cromatina/patologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Histonas/metabolismo , Humanos , Lisina/metabolismo , Modelos Moleculares , Complexos Multienzimáticos , Ressonância Magnética Nuclear Biomolecular , Nucleossomos/genética , Nucleossomos/patologia , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
P53-binding protein 1 (53BP1) is a multi-functional double-strand break repair protein that is essential for class switch recombination in B lymphocytes and for sensitizing BRCA1-deficient tumours to poly-ADP-ribose polymerase-1 (PARP) inhibitors. Central to all 53BP1 activities is its recruitment to double-strand breaks via the interaction of the tandem Tudor domain with dimethylated lysine 20 of histone H4 (H4K20me2). Here we identify an uncharacterized protein, Tudor interacting repair regulator (TIRR), that directly binds the tandem Tudor domain and masks its H4K20me2 binding motif. Upon DNA damage, the protein kinase ataxia-telangiectasia mutated (ATM) phosphorylates 53BP1 and recruits RAP1-interacting factor 1 (RIF1) to dissociate the 53BP1-TIRR complex. However, overexpression of TIRR impedes 53BP1 function by blocking its localization to double-strand breaks. Depletion of TIRR destabilizes 53BP1 in the nuclear-soluble fraction and alters the double-strand break-induced protein complex centring 53BP1. These findings identify TIRR as a new factor that influences double-strand break repair using a unique mechanism of masking the histone methyl-lysine binding function of 53BP1.
Assuntos
Proteínas de Transporte/metabolismo , Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sítios de Ligação , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Feminino , Humanos , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Ligação Proteica , Domínios Proteicos , Proteínas de Ligação a RNA , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/químicaRESUMO
The translesion synthesis (TLS) DNA polymerases Rev1 and Polζ function together in DNA lesion bypass during DNA replication, acting as nucleotide inserter and extender polymerases, respectively. While the structural characterization of the Saccharomyces cerevisiae Polζ in its DNA-bound state has illuminated how this enzyme synthesizes DNA, a mechanistic understanding of TLS also requires probing conformational changes associated with DNA- and Rev1 binding. Here, we used single-particle cryo-electron microscopy to determine the structure of the apo Polζ holoenzyme. We show that compared with its DNA-bound state, apo Polζ displays enhanced flexibility that correlates with concerted motions associated with expansion of the Polζ DNA-binding channel upon DNA binding. We also identified a lysine residue that obstructs the DNA-binding channel in apo Polζ, suggesting a gating mechanism. The Polζ subunit Rev7 is a hub protein that directly binds Rev1 and is a component of several other protein complexes such as the shieldin DNA double-strand break repair complex. We analyzed the molecular interactions of budding yeast Rev7 in the context of Polζ and those of human Rev7 in the context of shieldin using a crystal structure of Rev7 bound to a fragment of the shieldin-3 protein. Overall, our study provides new insights into Polζ mechanism of action and the manner in which Rev7 recognizes partner proteins.
Assuntos
Microscopia Crioeletrônica/métodos , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Nucleotidiltransferases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , DNA Polimerase Dirigida por DNA/química , Humanos , Conformação ProteicaRESUMO
Dynamic variations in the structure of chromatin influence virtually all DNA-related processes in eukaryotes and are controlled in part by post-translational modifications of histones. One such modification, the acetylation of lysine 56 (H3K56ac) in the amino-terminal α-helix (αN) of histone H3, has been implicated in the regulation of nucleosome assembly during DNA replication and repair, and nucleosome disassembly during gene transcription. In Saccharomyces cerevisiae, the histone chaperone Rtt106 contributes to the deposition of newly synthesized H3K56ac-carrying H3-H4 complex on replicating DNA, but it is unclear how Rtt106 binds H3-H4 and specifically recognizes H3K56ac as there is no apparent acetylated lysine reader domain in Rtt106. Here, we show that two domains of Rtt106 are involved in a combinatorial recognition of H3-H4. An N-terminal domain homodimerizes and interacts with H3-H4 independently of acetylation while a double pleckstrin-homology (PH) domain binds the K56-containing region of H3. Affinity is markedly enhanced upon acetylation of K56, an effect that is probably due to increased conformational entropy of the αN helix of H3. Our data support a mode of interaction where the N-terminal homodimeric domain of Rtt106 intercalates between the two H3-H4 components of the (H3-H4)(2) tetramer while two double PH domains in the Rtt106 dimer interact with each of the two H3K56ac sites in (H3-H4)(2). We show that the Rtt106-(H3-H4)(2) interaction is important for gene silencing and the DNA damage response.
Assuntos
Histonas/química , Histonas/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/química , Acetilação , Animais , Sítios de Ligação , Cristalografia por Raios X , Dano ao DNA , Inativação Gênica , Instabilidade Genômica , Lisina/análogos & derivados , Lisina/química , Lisina/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Chaperonas Moleculares/genética , Mutação/genética , Maleabilidade , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/genética , Relação Estrutura-Atividade , Especificidade por Substrato , Xenopus laevisRESUMO
We report a broader than previously appreciated clinical spectrum for hereditary sensory and autonomic neuropathy type 1E (HSAN1E) and a potential pathogenic mechanism for DNA methyltransferase (DNMT1) mutations. The clinical presentations and genetic characteristics of nine newly identified HSAN1E kinships (45 affected subjects) were investigated. Five novel mutations of DNMT1 were discovered; p.C353F, p.T481P, p.P491L, p.Y524D and p.I531N, all within the target-sequence domain, and two mutations (p.T481P, p.P491L) arising de novo. Recently, HSAN1E has been suggested as an allelic disorder of autosomal dominant cerebellar ataxia, deafness and narcolepsy. Our results indicate that all the mutations causal for HSAN1E are located in the middle part or N-terminus end of the TS domain, whereas all the mutations causal for autosomal dominant cerebellar ataxia, deafness and narcolepsy are located in the C-terminus end of the TS domain. The impact of the seven causal mutations in this cohort was studied by cellular localization experiments. The binding efficiency of the mutant DNMT proteins at the replication foci and heterochromatin were evaluated. Phenotypic characterizations included electromyography, brain magnetic resonance and nuclear imaging, electroencephalography, sural nerve biopsies, sleep evaluation and neuropsychometric testing. The average survival of HSAN1E was 53.6 years. [standard deviation = 7.7, range 43-75 years], and mean onset age was 37.7 years. (standard deviation = 8.6, range 18-51 years). Expanded phenotypes include myoclonic seizures, auditory or visual hallucinations, and renal failure. Hypersomnia, rapid eye movement sleep disorder and/or narcolepsy were identified in 11 subjects. Global brain atrophy was found in 12 of 14 who had brain MRI. EEGs showed low frequency (delta waves) frontal-predominant abnormality in five of six patients. Marked variability in cognitive deficits was observed, but the majority of patients (89%) developed significant cognitive deficit by the age of 45 years. Cognitive function decline often started with personality changes and psychiatric manifestations. A triad of hearing loss, sensory neuropathy and cognitive decline remains as the stereotypic presentation of HSAN1E. Moreover, we show that mutant DNMT1 proteins translocate to the cytoplasm and are prone to form aggresomes while losing their binding ability to heterochromatin during the G2 cell cycle. Our results suggest mutations in DNMT1 result in imbalanced protein homeostasis through aggresome-induced autophagy. This mechanism may explain why mutations in the sole DNA maintenance methyltransferase lead to selective central and peripheral neurodegeneration.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Neuropatias Hereditárias Sensoriais e Autônomas/diagnóstico , Neuropatias Hereditárias Sensoriais e Autônomas/genética , Mutação/genética , Adulto , Idoso , Autofagia/genética , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/química , Feminino , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Doenças do Sistema Nervoso/diagnóstico , Doenças do Sistema Nervoso/genética , Linhagem , Estrutura Secundária de ProteínaRESUMO
Tyr142, the C-terminal amino acid of histone variant H2A.X is phosphorylated by WSTF (Williams-Beuren syndrome transcription factor), a component of the WICH complex (WSTF-ISWI chromatin-remodeling complex), under basal conditions in the cell. In response to DNA double-strand breaks (DSBs), H2A.X is instantaneously phosphorylated at Ser139 by the kinases ATM and ATR and is progressively dephosphorylated at Tyr142 by the Eya1 and Eya3 tyrosine phosphatases, resulting in a temporal switch from a postulated diphosphorylated (pSer139, pTyr142) to monophosphorylated (pSer139) H2A.X state. How mediator proteins interpret these two signals remains a question of fundamental interest. We provide structural, biochemical, and cellular evidence that Microcephalin (MCPH1), an early DNA damage response protein, can read both modifications via its tandem BRCA1 C-terminal (BRCT) domains, thereby emerging as a versatile sensor of H2A.X phosphorylation marks. We show that MCPH1 recruitment to sites of DNA damage is linked to both states of H2A.X.
Assuntos
Reparo do DNA/fisiologia , Histonas/metabolismo , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Fosfosserina/metabolismo , Fosfotirosina/metabolismo , Calorimetria , Proteínas de Ciclo Celular , Clonagem Molecular , Cristalografia por Raios X , Proteínas do Citoesqueleto , Dano ao DNA/fisiologia , Escherichia coli , Vetores Genéticos/genética , Humanos , Microscopia de Fluorescência , Proteínas do Tecido Nervoso/genéticaRESUMO
FAM111A, a serine protease, plays roles in DNA replication and antiviral defense. Missense mutations in the catalytic domain cause hyper-autocleavage and are associated with genetic disorders with developmental defects. Despite the enzyme's biological significance, the molecular architecture of the FAM111A serine protease domain (SPD) is unknown. Here, we show that FAM111A is a dimerization-dependent protease containing a narrow, recessed active site that cleaves substrates with a chymotrypsin-like specificity. X-ray crystal structures and mutagenesis studies reveal that FAM111A dimerizes via the N-terminal helix within the SPD. This dimerization induces an activation cascade from the dimerization sensor loop to the oxyanion hole through disorder-to-order transitions. Dimerization is essential for proteolytic activity in vitro and for facilitating DNA replication at DNA-protein crosslink obstacles in cells, while it is dispensable for autocleavage. These findings underscore the role of dimerization in FAM111A's function and highlight the distinction in its dimerization dependency between substrate cleavage and autocleavage.
Assuntos
Serina Endopeptidases , Serina Proteases , Dimerização , Serina Endopeptidases/metabolismo , Proteólise , Replicação do DNA , SerinaRESUMO
The characterization of short-lived intermediate states along the path of a ubiquitylation reaction remains a major challenge. In this issue of Chem, Ai et al. present a chemical trapping method for probing transient intermediates in substrate ubiquitylation. The usefulness of this approach is demonstrated by the determination of single-particle cryo-EM structures related to nucleosome ubiquitylation.
RESUMO
The recruitment of 53BP1 to chromatin, mediated by its recognition of histone H4 dimethylated at lysine 20 (H4K20me2), is important for DNA double-strand break repair. Using a series of small molecule antagonists, we demonstrate a conformational equilibrium between an open and a pre-existing lowly populated closed state of 53BP1 in which the H4K20me2 binding surface is buried at the interface between two interacting 53BP1 molecules. In cells, these antagonists inhibit the chromatin recruitment of wild type 53BP1, but do not affect 53BP1 variants unable to access the closed conformation despite preservation of the H4K20me2 binding site. Thus, this inhibition operates by shifting the conformational equilibrium toward the closed state. Our work therefore identifies an auto-associated form of 53BP1-autoinhibited for chromatin binding-that can be stabilized by small molecule ligands encapsulated between two 53BP1 protomers. Such ligands are valuable research tools to study the function of 53BP1 and have the potential to facilitate the development of new drugs for cancer therapy.
Assuntos
Cromatina , Histonas , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Histonas/metabolismo , Engenharia de Proteínas , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , HumanosRESUMO
The recruitment of 53BP1 to chromatin, mediated by its recognition of histone H4 dimethylated at lysine 20 (H4K20me2), is important for DNA double-strand break repair. Using a series of small molecule antagonists, we demonstrate a conformational equilibrium between an open and a pre-existing lowly populated closed state of 53BP1 in which the H4K20me2 binding surface is buried at the interface between two interacting 53BP1 molecules. In cells, these antagonists inhibit the chromatin recruitment of wild type 53BP1, but do not affect 53BP1 variants unable to access the closed conformation despite preservation of the H4K20me2 binding site. Thus, this inhibition operates by shifting the conformational equilibrium toward the closed state. Our work therefore identifies an auto-associated form of 53BP1 - autoinhibited for chromatin binding - that can be stabilized by small molecule ligands encapsulated between two 53BP1 protomers. Such ligands are valuable research tools to study the function of 53BP1 and have the potential to facilitate the development of new drugs for cancer therapy.
RESUMO
The histone chaperone FACT (facilitates chromatin transcription) enhances transcription in eukaryotic cells, targeting DNA-protein interactions. FACT, a heterodimer in humans, comprises SPT16 and SSRP1 subunits. We measure nucleosome stability and dynamics in the presence of FACT and critical component domains. Optical tweezers quantify FACT/subdomain binding to nucleosomes, displacing the outer wrap of DNA, disrupting direct DNA-histone (core site) interactions, altering the energy landscape of unwrapping, and increasing the kinetics of DNA-histone disruption. Atomic force microscopy reveals nucleosome remodeling, while single-molecule fluorescence quantifies kinetics of histone loss for disrupted nucleosomes, a process accelerated by FACT. Furthermore, two isolated domains exhibit contradictory functions; while the SSRP1 HMGB domain displaces DNA, SPT16 MD/CTD stabilizes DNA-H2A/H2B dimer interactions. However, only intact FACT tethers disrupted DNA to the histones and supports rapid nucleosome reformation over several cycles of force disruption/release. These results demonstrate that key FACT domains combine to catalyze both nucleosome disassembly and reassembly.
Assuntos
Chaperonas de Histonas , Nucleossomos , Humanos , Cromatina , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Fatores de Elongação da Transcrição/genéticaRESUMO
Hsp90 is an ATP-dependent molecular chaperone. The best characterized inhibitors of Hsp90 target its ATP binding pocket, causing nonselective degradation of Hsp90 client proteins. Here, we show that the small molecule celastrol inhibits the Hsp90 chaperoning machinery by inactivating the co-chaperone p23, resulting in a more selective destabilization of steroid receptors compared with kinase clients. Our in vitro and in vivo results demonstrate that celastrol disrupts p23 function by altering its three-dimensional structure, leading to rapid formation of amyloid-like fibrils. This study reveals a unique inhibition mechanism of p23 by a small molecule that could be exploited in the dissection of protein fibrillization processes as well as in the therapeutics of steroid receptor-dependent diseases.
Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Oxirredutases Intramoleculares/metabolismo , Chaperonas Moleculares/metabolismo , Receptores de Esteroides/metabolismo , Triterpenos/farmacologia , Trifosfato de Adenosina/metabolismo , Amiloide/química , Amiloide/metabolismo , Amiloide/ultraestrutura , Animais , Western Blotting , Linhagem Celular , Proteínas de Choque Térmico HSP90/genética , Células HeLa , Humanos , Oxirredutases Intramoleculares/química , Oxirredutases Intramoleculares/genética , Espectroscopia de Ressonância Magnética , Microscopia Imunoeletrônica , Modelos Moleculares , Chaperonas Moleculares/genética , Mutagênese Sítio-Dirigida , Triterpenos Pentacíclicos , Prostaglandina-E Sintases , Ligação Proteica/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Interferência de RNA , Receptores de Progesterona/metabolismo , Proteínas Recombinantes/metabolismo , SpodopteraRESUMO
Geminin and its binding partner Cdt1 are essential for the regulation of DNA replication. Here we show that the CULLIN3 E3 ubiquitin ligase adaptor protein SPOP binds Geminin at endogenous level and regulates DNA replication. SPOP promotes K27-linked non-degradative poly-ubiquitination of Geminin at lysine residues 100 and 127. This poly-ubiquitination of Geminin prevents DNA replication over-firing by indirectly blocking the association of Cdt1 with the MCM protein complex, an interaction required for DNA unwinding and replication. SPOP is frequently mutated in certain human cancer types and implicated in tumorigenesis. We show that cancer-associated SPOP mutations impair Geminin K27-linked poly-ubiquitination and induce replication origin over-firing and re-replication. The replication stress caused by SPOP mutations triggers replication catastrophe and cell death upon ATR inhibition. Our results reveal a tumor suppressor role of SPOP in preventing DNA replication over-firing and genome instability and suggest that SPOP-mutated tumors may be susceptible to ATR inhibitor therapy.
Assuntos
Geminina/metabolismo , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Complexos Ubiquitina-Proteína Ligase/genética , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Replicação do DNA/genética , Replicação do DNA/fisiologia , Geminina/genética , Humanos , Masculino , Camundongos , Camundongos SCID , Proteínas de Manutenção de Minicromossomo/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Mutação/genética , Ubiquitinação/genética , Ubiquitinação/fisiologiaRESUMO
53BP1 activates nonhomologous end joining (NHEJ) and inhibits homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Dissociation of 53BP1 from DSBs and consequent activation of HR, a less error-prone pathway than NHEJ, helps maintain genome integrity during DNA replication; however, the underlying mechanisms are not fully understood. Here, we demonstrate that E3 ubiquitin ligase SPOP promotes HR during S phase of the cell cycle by excluding 53BP1 from DSBs. In response to DNA damage, ATM kinase-catalyzed phosphorylation of SPOP causes a conformational change in SPOP, revealed by x-ray crystal structures, that stabilizes its interaction with 53BP1. 53BP1-bound SPOP induces polyubiquitination of 53BP1, eliciting 53BP1 extraction from chromatin by a valosin-containing protein/p97 segregase complex. Our work shows that SPOP facilitates HR repair over NHEJ during DNA replication by contributing to 53BP1 removal from chromatin. Cancer-derived SPOP mutations block SPOP interaction with 53BP1, inducing HR defects and chromosomal instability.