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1.
Mol Cell ; 73(3): 621-638.e17, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30554943

RESUMO

Targeting bromodomains (BRDs) of the bromo-and-extra-terminal (BET) family offers opportunities for therapeutic intervention in cancer and other diseases. Here, we profile the interactomes of BRD2, BRD3, BRD4, and BRDT following treatment with the pan-BET BRD inhibitor JQ1, revealing broad rewiring of the interaction landscape, with three distinct classes of behavior for the 603 unique interactors identified. A group of proteins associate in a JQ1-sensitive manner with BET BRDs through canonical and new binding modes, while two classes of extra-terminal (ET)-domain binding motifs mediate acetylation-independent interactions. Last, we identify an unexpected increase in several interactions following JQ1 treatment that define negative functions for BRD3 in the regulation of rRNA synthesis and potentially RNAPII-dependent gene expression that result in decreased cell proliferation. Together, our data highlight the contributions of BET protein modules to their interactomes allowing for a better understanding of pharmacological rewiring in response to JQ1.


Assuntos
Antineoplásicos/farmacologia , Azepinas/farmacologia , Terapia de Alvo Molecular/métodos , Neoplasias/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas de Ligação a RNA/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Triazóis/farmacologia , Antineoplásicos/química , Azepinas/química , Proteínas de Ciclo Celular , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Células K562 , Modelos Moleculares , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica/métodos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Triazóis/química
2.
Nucleic Acids Res ; 51(1): 475-487, 2023 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-36546776

RESUMO

NSP14 is a dual function enzyme containing an N-terminal exonuclease domain (ExoN) and C-terminal Guanine-N7-methyltransferase (N7-MTase) domain. Both activities are essential for the viral life cycle and may be targeted for anti-viral therapeutics. NSP14 forms a complex with NSP10, and this interaction enhances the nuclease but not the methyltransferase activity. We have determined the structure of SARS-CoV-2 NSP14 in the absence of NSP10 to 1.7 Å resolution. Comparisons with NSP14/NSP10 complexes reveal significant conformational changes that occur within the NSP14 ExoN domain upon binding of NSP10, including helix to coil transitions that facilitate the formation of the ExoN active site and provide an explanation of the stimulation of nuclease activity by NSP10. We have determined the structure of NSP14 in complex with cap analogue 7MeGpppG, and observe conformational changes within a SAM/SAH interacting loop that plays a key role in viral mRNA capping offering new insights into MTase activity. We perform an X-ray fragment screen on NSP14, revealing 72 hits bound to sites of inhibition in the ExoN and MTase domains. These fragments serve as excellent starting point tools for structure guided development of NSP14 inhibitors that may be used to treat COVID-19 and potentially other future viral threats.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , RNA Mensageiro , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Antivirais/farmacologia , Exorribonucleases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Metiltransferases/metabolismo , RNA Viral/genética , RNA Viral/metabolismo
3.
Nucleic Acids Res ; 51(18): 9920-9937, 2023 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-37665033

RESUMO

Polymerase theta (Polθ) acts in DNA replication and repair, and its inhibition is synthetic lethal in BRCA1 and BRCA2-deficient tumor cells. Novobiocin (NVB) is a first-in-class inhibitor of the Polθ ATPase activity, and it is currently being tested in clinical trials as an anti-cancer drug. Here, we investigated the molecular mechanism of NVB-mediated Polθ inhibition. Using hydrogen deuterium exchange-mass spectrometry (HX-MS), biophysical, biochemical, computational and cellular assays, we found NVB is a non-competitive inhibitor of ATP hydrolysis. NVB sugar group deletion resulted in decreased potency and reduced HX-MS interactions, supporting a specific NVB binding orientation. Collective results revealed that NVB binds to an allosteric site to block DNA binding, both in vitro and in cells. Comparisons of The Cancer Genome Atlas (TCGA) tumors and matched controls implied that POLQ upregulation in tumors stems from its role in replication stress responses to increased cell proliferation: this can now be tested in fifteen tumor types by NVB blocking ssDNA-stimulation of ATPase activity, required for Polθ function at replication forks and DNA damage sites. Structural and functional insights provided in this study suggest a path for developing NVB derivatives with improved potency for Polθ inhibition by targeting ssDNA binding with entropically constrained small molecules.


Assuntos
Adenosina Trifosfatases , DNA Polimerase teta , Neoplasias , Novobiocina , Humanos , Adenosina Trifosfatases/metabolismo , Replicação do DNA , DNA de Cadeia Simples , DNA Polimerase Dirigida por DNA/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Novobiocina/farmacologia
4.
Mol Cell ; 64(4): 704-719, 2016 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-27871366

RESUMO

The cytotoxicity of DNA-protein crosslinks (DPCs) is largely ascribed to their ability to block the progression of DNA replication. DPCs frequently occur in cells, either as a consequence of metabolism or exogenous agents, but the mechanism of DPC repair is not completely understood. Here, we characterize SPRTN as a specialized DNA-dependent and DNA replication-coupled metalloprotease for DPC repair. SPRTN cleaves various DNA binding substrates during S-phase progression and thus protects proliferative cells from DPC toxicity. Ruijs-Aalfs syndrome (RJALS) patient cells with monogenic and biallelic mutations in SPRTN are hypersensitive to DPC-inducing agents due to a defect in DNA replication fork progression and the inability to eliminate DPCs. We propose that SPRTN protease represents a specialized DNA replication-coupled DPC repair pathway essential for DNA replication progression and genome stability. Defective SPRTN-dependent clearance of DPCs is the molecular mechanism underlying RJALS, and DPCs are contributing to accelerated aging and cancer.


Assuntos
Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , DNA/química , Instabilidade Genômica , Sequência de Aminoácidos , Sítios de Ligação , Reagentes de Ligações Cruzadas/química , DNA/genética , DNA/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/genética , Etoposídeo/química , Formaldeído/química , Expressão Gênica , Humanos , Cinética , Mutação , Ligação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Síndrome , Raios Ultravioleta
5.
Nucleic Acids Res ; 49(16): 9294-9309, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34387694

RESUMO

The SNM1 nucleases which help maintain genome integrity are members of the metallo-ß-lactamase (MBL) structural superfamily. Their conserved MBL-ß-CASP-fold SNM1 core provides a molecular scaffold forming an active site which coordinates the metal ions required for catalysis. The features that determine SNM1 endo- versus exonuclease activity, and which control substrate selectivity and binding are poorly understood. We describe a structure of SNM1B/Apollo with two nucleotides bound to its active site, resembling the product state of its exonuclease reaction. The structure enables definition of key SNM1B residues that form contacts with DNA and identifies a 5' phosphate binding pocket, which we demonstrate is important in catalysis and which has a key role in determining endo- versus exonucleolytic activity across the SNM1 family. We probed the capacity of SNM1B to digest past sites of common endogenous DNA lesions and find that base modifications planar to the nucleobase can be accommodated due to the open architecture of the active site, but lesions axial to the plane of the nucleobase are not well tolerated due to constriction around the altered base. We propose that SNM1B/Apollo might employ its activity to help remove common oxidative lesions from telomeres.


Assuntos
Endonucleases/química , Exodesoxirribonucleases/química , Exonucleases/química , beta-Lactamases/genética , Sítios de Ligação/genética , Catálise , Domínio Catalítico/genética , Proteínas de Ligação a DNA , Endonucleases/genética , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/ultraestrutura , Exonucleases/genética , Humanos , Metais , Fosfatos/química , beta-Lactamases/química
6.
Nucleic Acids Res ; 49(16): 9310-9326, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34387696

RESUMO

Artemis (SNM1C/DCLRE1C) is an endonuclease that plays a key role in development of B- and T-lymphocytes and in dsDNA break repair by non-homologous end-joining (NHEJ). Artemis is phosphorylated by DNA-PKcs and acts to open DNA hairpin intermediates generated during V(D)J and class-switch recombination. Artemis deficiency leads to congenital radiosensitive severe acquired immune deficiency (RS-SCID). Artemis belongs to a superfamily of nucleases containing metallo-ß-lactamase (MBL) and ß-CASP (CPSF-Artemis-SNM1-Pso2) domains. We present crystal structures of the catalytic domain of wildtype and variant forms of Artemis, including one causing RS-SCID Omenn syndrome. The catalytic domain of the Artemis has similar endonuclease activity to the phosphorylated full-length protein. Our structures help explain the predominantly endonucleolytic activity of Artemis, which contrasts with the predominantly exonuclease activity of the closely related SNM1A and SNM1B MBL fold nucleases. The structures reveal a second metal binding site in its ß-CASP domain unique to Artemis, which is amenable to inhibition by compounds including ebselen. By combining our structural data with that from a recently reported Artemis structure, we were able model the interaction of Artemis with DNA substrates. The structures, including one of Artemis with the cephalosporin ceftriaxone, will help enable the rational development of selective SNM1 nuclease inhibitors.


Assuntos
Proteínas de Ciclo Celular/ultraestrutura , Proteínas de Ligação a DNA/ultraestrutura , Endonucleases/ultraestrutura , Exodesoxirribonucleases/ultraestrutura , Imunodeficiência Combinada Severa/genética , Linfócitos B/enzimologia , Domínio Catalítico/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cristalografia por Raios X , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Endonucleases/antagonistas & inibidores , Endonucleases/química , Endonucleases/genética , Inibidores Enzimáticos/química , Exodesoxirribonucleases/química , Exodesoxirribonucleases/genética , Humanos , Fosforilação/genética , Dobramento de Proteína , Imunodeficiência Combinada Severa/enzimologia , Imunodeficiência Combinada Severa/patologia , Linfócitos T/enzimologia
7.
J Biol Chem ; 295(10): 2948-2958, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-31914405

RESUMO

Forkhead box N1 (FOXN1) is a member of the forkhead box family of transcription factors and plays an important role in thymic epithelial cell differentiation and development. FOXN1 mutations in humans and mice give rise to the "nude" phenotype, which is marked by athymia. FOXN1 belongs to a subset of the FOX family that recognizes an alternative forkhead-like (FHL) consensus sequence (GACGC) that is different from the more widely recognized forkhead (FKH) sequence RYAAAYA (where R is purine, and Y is pyrimidine). Here, we present the FOXN1 structure in complex with DNA containing an FHL motif at 1.6 Å resolution, in which the DNA sequence is recognized by a mixture of direct and water-mediated contacts provided by residues in an α-helix inserted in the DNA major groove (the recognition helix). Comparisons with the structure of other FOX family members revealed that the FKH and FHL DNA sequences are bound in two distinct modes, with partially different registers for the protein DNA contacts. We identified a single alternative rotamer within the recognition helix itself as an important determinant of DNA specificity and found protein sequence features in the recognition helix that could be used to predict the specificity of other FOX family members. Finally, we demonstrate that the C-terminal region of FOXN1 is required for high-affinity DNA binding and that FOXN1 has a significantly reduced affinity for DNA that contains 5'-methylcytosine, which may have implications for the role of FOXN1 in thymic involution.


Assuntos
DNA/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Cristalografia por Raios X , DNA/química , Metilação de DNA , Ensaio de Desvio de Mobilidade Eletroforética , Fatores de Transcrição Forkhead/química , Fatores de Transcrição Forkhead/genética , Humanos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência
8.
Hum Mutat ; 40(5): 566-577, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30817846

RESUMO

There is still around 50% of the familial breast cancer (BC) cases with an undefined genetic cause, here we have used next-generation sequencing (NGS) technology to identify new BC susceptibility genes. This approach has led to the identification of RECQL5, a member of RECQL-helicases family, as a new BC susceptibility candidate, which deserves further study. We have used a combination of whole exome sequencing in a family negative for mutations in BRCA1/2 throughout (BRCAX), in which we found a probably deleterious variant in RECQL5, and targeted NGS of the complete coding regions and exon-intron boundaries of the candidate gene in 699 BC Spanish BRCAX families and 665 controls. Functional characterization and in silico inference of pathogenicity were performed to evaluate the deleterious effect of detected variants. We found at least seven deleterious or likely deleterious variants among the cases and only one in controls. These results prompt us to propose RECQL5 as a gene that would be worth to analyze in larger studies to explore its possible implication in BC susceptibility.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Predisposição Genética para Doença , RecQ Helicases/genética , RecQ Helicases/metabolismo , Processamento Alternativo , Proteína BRCA1/genética , Proteína BRCA2/genética , Biomarcadores Tumorais , Neoplasias da Mama/patologia , Biologia Computacional/métodos , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética , Variação Genética , Humanos , Perda de Heterozigosidade , Família Multigênica , Linhagem , Sequenciamento do Exoma
9.
J Biol Chem ; 293(33): 12862-12876, 2018 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-29880640

RESUMO

Mitochondrial tRNAs are transcribed as long polycistronic transcripts of precursor tRNAs and undergo posttranscriptional modifications such as endonucleolytic processing and methylation required for their correct structure and function. Among them, 5'-end processing and purine 9 N1-methylation of mitochondrial tRNA are catalyzed by two proteinaceous complexes with overlapping subunit composition. The Mg2+-dependent RNase P complex for 5'-end cleavage comprises the methyltransferase domain-containing protein tRNA methyltransferase 10C, mitochondrial RNase P subunit (TRMT10C/MRPP1), short-chain oxidoreductase hydroxysteroid 17ß-dehydrogenase 10 (HSD17B10/MRPP2), and metallonuclease KIAA0391/MRPP3. An MRPP1-MRPP2 subcomplex also catalyzes the formation of 1-methyladenosine/1-methylguanosine at position 9 using S-adenosyl-l-methionine as methyl donor. However, a lack of structural information has precluded insights into how these complexes methylate and process mitochondrial tRNA. Here, we used a combination of X-ray crystallography, interaction and activity assays, and small angle X-ray scattering (SAXS) to gain structural insight into the two tRNA modification complexes and their components. The MRPP1 N terminus is involved in tRNA binding and monomer-monomer self-interaction, whereas the C-terminal SPOUT fold contains key residues for S-adenosyl-l-methionine binding and N1-methylation. The entirety of MRPP1 interacts with MRPP2 to form the N1-methylation complex, whereas the MRPP1-MRPP2-MRPP3 RNase P complex only assembles in the presence of precursor tRNA. This study proposes low-resolution models of the MRPP1-MRPP2 and MRPP1-MRPP2-MRPP3 complexes that suggest the overall architecture, stoichiometry, and orientation of subunits and tRNA substrates.


Assuntos
3-Hidroxiacil-CoA Desidrogenases/química , Metiltransferases/química , Modelos Moleculares , Complexos Multienzimáticos/química , RNA Mitocondrial/química , RNA de Transferência/química , Ribonuclease P/química , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Cristalografia por Raios X , Humanos , Metiltransferases/metabolismo , Complexos Multienzimáticos/metabolismo , RNA Mitocondrial/metabolismo , RNA de Transferência/metabolismo , Ribonuclease P/metabolismo , Espalhamento a Baixo Ângulo
10.
Nucleic Acids Res ; 45(7): 4231-4243, 2017 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-28100692

RESUMO

RecQ helicases are important maintainers of genome integrity with distinct roles in almost every cellular process requiring access to DNA. RECQL5 is one of five human RecQ proteins and is particularly versatile in this regard, forming protein complexes with a diverse set of cellular partners in order to coordinate its helicase activity to various processes including replication, recombination and DNA repair. In this study, we have determined crystal structures of the core helicase domain of RECQL5 both with and without the nucleotide ADP in two distinctly different ('Open' and 'Closed') conformations. Small angle X-ray scattering studies show that the 'Open' form of the protein predominates in solution and we discuss implications of this with regards to the RECQL5 mechanism and conformational changes. We have measured the ATPase, helicase and DNA binding properties of various RECQL5 constructs and variants and discuss the role of these regions and residues in the various RECQL5 activities. Finally, we have performed a systematic comparison of the RECQL5 structures with other RecQ family structures and based on these comparisons we have constructed a model for the mechano-chemical cycle of the common catalytic core of these helicases.


Assuntos
Modelos Moleculares , RecQ Helicases/química , Domínio Catalítico , Humanos , Mutação , Conformação Proteica em alfa-Hélice , RecQ Helicases/genética , RecQ Helicases/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios X
11.
Nucleic Acids Res ; 43(22): 11047-60, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26582912

RESUMO

The human SNM1A and SNM1B/Apollo proteins are members of an extended family of eukaryotic nuclease containing a motif related to the prokaryotic metallo-ß-lactamase (MBL) fold. SNM1A is a key exonuclease during replication-dependent and transcription-coupled interstrand crosslink repair, while SNM1B/Apollo is required for maintaining telomeric overhangs. Here, we report the crystal structures of SNM1A and SNM1B at 2.16 Å. While both proteins contain a typical MBL-ß-CASP domain, a region of positive charge surrounds the active site of SNM1A, which is absent in SNM1B and explains the greater apparent processivity of SNM1A. The structures of both proteins also reveal a putative, wide DNA-binding groove. Extensive mutagenesis of this groove, coupled with detailed biochemical analysis, identified residues that did not impact on SNM1A catalytic activity, but drastically reduced its processivity. Moreover, we identified a key role for this groove for efficient digestion past DNA interstrand crosslinks, facilitating the key DNA repair reaction catalysed by SNM1A. Together, the architecture and dimensions of this groove, coupled to the surrounding region of high positive charge, explain the remarkable ability of SNM1A to accommodate and efficiently digest highly distorted DNA substrates, such as those containing DNA lesions.


Assuntos
Enzimas Reparadoras do DNA/química , DNA/metabolismo , Exodesoxirribonucleases/química , Proteínas Nucleares/química , Domínio Catalítico , Proteínas de Ciclo Celular , Dano ao DNA , Enzimas Reparadoras do DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Humanos , Modelos Moleculares , Proteínas Nucleares/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína
12.
Nucleic Acids Res ; 43(10): 5221-35, 2015 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-25901030

RESUMO

Bloom's syndrome helicase (BLM) is a member of the RecQ family of DNA helicases, which play key roles in the maintenance of genome integrity in all organism groups. We describe crystal structures of the BLM helicase domain in complex with DNA and with an antibody fragment, as well as SAXS and domain association studies in solution. We show an unexpected nucleotide-dependent interaction of the core helicase domain with the conserved, poorly characterized HRDC domain. The BLM-DNA complex shows an unusual base-flipping mechanism with unique positioning of the DNA duplex relative to the helicase core domains. Comparison with other crystal structures of RecQ helicases permits the definition of structural transitions underlying ATP-driven helicase action, and the identification of a nucleotide-regulated tunnel that may play a role in interactions with complex DNA substrates.


Assuntos
RecQ Helicases/química , Difosfato de Adenosina/química , Cristalografia por Raios X , DNA/química , Modelos Moleculares , Mutação , Conformação Proteica , Estrutura Terciária de Proteína , RecQ Helicases/genética , RecQ Helicases/metabolismo , Anticorpos de Domínio Único/química , Zinco/química
13.
J Biol Chem ; 290(13): 8539-49, 2015 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-25670864

RESUMO

Ets-2, like its closely related homologue Ets-1, is a member of the Ets family of DNA binding transcription factors. Both proteins are subject to multiple levels of regulation of their DNA binding and transactivation properties. One such regulatory mechanism is the presence of an autoinhibitory module, which in Ets-1 allosterically inhibits the DNA binding activity. This inhibition can be relieved by interaction with protein partners or cooperative binding to closely separated Ets binding sites in a palindromic arrangement. In this study we describe the 2.5 Å resolution crystal structure of a DNA complex of the Ets-2 Ets domain. The Ets domain crystallized with two distinct species in the asymmetric unit, which closely resemble the autoinhibited and DNA bound forms of Ets-1. This discovery prompted us to re-evaluate the current model for the autoinhibitory mechanism and the structural basis for cooperative DNA binding. In contrast to Ets-1, in which the autoinhibition is caused by a combination of allosteric and steric mechanisms, we were unable to find clear evidence for the allosteric mechanism in Ets-2. We also demonstrated two possibly distinct types of cooperative binding to substrates with Ets binding motifs separated by four and six base pairs and suggest possible molecular mechanisms for this behavior.


Assuntos
Proteína Proto-Oncogênica c-ets-2/química , Regulação Alostérica , Cristalografia por Raios X , DNA/química , Humanos , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
14.
J Biol Chem ; 290(22): 13692-709, 2015 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-25866208

RESUMO

Ets transcription factors, which share the conserved Ets DNA-binding domain, number nearly 30 members in humans and are particularly involved in developmental processes. Their deregulation following changes in expression, transcriptional activity, or by chromosomal translocation plays a critical role in carcinogenesis. Ets DNA binding, selectivity, and regulation have been extensively studied; however, questions still arise regarding binding specificity outside the core GGA recognition sequence and the mode of action of Ets post-translational modifications. Here, we report the crystal structures of Etv1, Etv4, Etv5, and Fev, alone and in complex with DNA. We identify previously unrecognized features of the protein-DNA interface. Interactions with the DNA backbone account for most of the binding affinity. We describe a highly coordinated network of water molecules acting in base selection upstream of the GGAA core and the structural features that may account for discrimination against methylated cytidine residues. Unexpectedly, all proteins crystallized as disulfide-linked dimers, exhibiting a novel interface (distant to the DNA recognition helix). Homodimers of Etv1, Etv4, and Etv5 could be reduced to monomers, leading to a 40-200-fold increase in DNA binding affinity. Hence, we present the first indication of a redox-dependent regulatory mechanism that may control the activity of this subset of oncogenic Ets transcription factors.


Assuntos
Proteínas E1A de Adenovirus/química , Proteínas de Ligação a DNA/química , Dissulfetos/química , Proteínas Nucleares/química , Proteínas Proto-Oncogênicas c-ets/química , Proteínas Proto-Oncogênicas/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Cromatografia Líquida , DNA/química , Regulação da Expressão Gênica , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Neoplasias/metabolismo , Oxirredução , Oxigênio/química , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por Electrospray
15.
J Bacteriol ; 196(2): 265-75, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24163345

RESUMO

Bacillus subtilis mutants lacking ymdB are unable to form biofilms, exhibit a strong overexpression of the flagellin gene hag, and are deficient in SlrR, a SinR antagonist. Here, we report the functional and structural characterization of YmdB, and we find that YmdB is a phosphodiesterase with activity against 2',3'- and 3',5'-cyclic nucleotide monophosphates. The structure of YmdB reveals that the enzyme adopts a conserved phosphodiesterase fold with a binuclear metal center. Mutagenesis of a catalytically crucial residue demonstrates that the enzymatic activity of YmdB is essential for biofilm formation. The deletion of ymdB affects the expression of more than 800 genes; the levels of the σ(D)-dependent motility regulon and several sporulation genes are increased, and the levels of the SinR-repressed biofilm genes are decreased, confirming the role of YmdB in regulating late adaptive responses of B. subtilis.


Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Regulação Bacteriana da Expressão Gênica , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Bacillus subtilis/fisiologia , Biofilmes/crescimento & desenvolvimento , Cristalografia por Raios X , Análise Mutacional de DNA , Deleção de Genes , Modelos Moleculares , Conformação Proteica
16.
J Biol Chem ; 288(15): 10766-78, 2013 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-23430750

RESUMO

Bacterial biofilms are complex communities of cells that are attached to a surface by an extracellular matrix. Biofilms are an increasing environmental and healthcare issue, causing problems ranging from the biofouling of ocean-going vessels, to dental plaque, infections of the urinary tract, and contamination of medical instruments such as catheters. A complete understanding of biofilm formation therefore requires knowledge of the regulatory pathways underpinning its formation so that effective intervention strategies can be determined. The master regulator that determines whether the gram-positive model organism Bacillus subtilis switches from a free-living, planktonic lifestyle to form a biofilm is called SinR. The activity of SinR, a transcriptional regulator, is controlled by its antagonists, SinI, SlrA, and SlrR. The interaction of these four proteins forms a switch, which determines whether or not SinR can inhibit biofilm formation by its repression of a number of extracellular matrix-associated operons. To determine the thermodynamic and kinetic parameters governing the protein-protein and protein-DNA interactions at the heart of this epigenetic switch, we have analyzed the protein-protein and protein-DNA interactions by isothermal titration calorimetry and surface plasmon resonance. We also present the crystal structure of SinR in complex with DNA, revealing the molecular basis of base-specific DNA recognition by SinR and suggesting that the most effective means of transcriptional control occurs by the looping of promoter DNA. The structural analysis also enables predictions about how SinR activity is controlled by its interaction with its antagonists.


Assuntos
Bacillus subtilis/fisiologia , Proteínas de Bactérias/metabolismo , Biofilmes , DNA Bacteriano/metabolismo , Regiões Promotoras Genéticas/fisiologia , Transcrição Gênica/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Ligação Proteica , Ressonância de Plasmônio de Superfície
17.
Biochem Soc Trans ; 42(1): 130-8, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24450640

RESUMO

The Ets family of eukaryotic transcription factors is based around the conserved Ets DNA-binding domain. Although their DNA-binding selectivity is biochemically and structurally well characterized, structures of homodimeric and ternary complexes point to Ets domains functioning as versatile protein-interaction modules. In the present paper, we review the progress made over the last decade to elucidate the structural mechanisms involved in modulation of DNA binding and protein partner selection during dimerization. We see that Ets domains, although conserved around a core architecture, have evolved to utilize a variety of interaction surfaces and binding mechanisms, reflecting Ets domains as dynamic interfaces for both DNA and protein interaction. Furthermore, we discuss recent advances in drug development for inhibition of Ets factors, and the roles structural biology can play in their future.


Assuntos
Neoplasias/genética , Proteínas Proto-Oncogênicas c-ets/química , Animais , Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Modelos Moleculares , Terapia de Alvo Molecular , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/química , Complexos Multiproteicos/fisiologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Proto-Oncogênicas c-ets/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-ets/fisiologia
18.
bioRxiv ; 2024 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-39282455

RESUMO

The SNM1A exonuclease plays a key role in repair of interstrand crosslinks (ICLs) which represent a particularly toxic class of DNA damage. Previous work suggests that the SWI/SNF family ATP-dependent, chromatin remodeler, Cockayne Syndrome B protein (CSB) interacts with SNM1A, during transcription-coupled DNA interstrand crosslink repair (TC-ICL repair). Here, we validate this interaction using purified proteins and demonstrate that the ubiquitin-binding and winged-helix domains of CSB are required for interaction with the catalytic domain of SNM1A. The winged helix domain is essential for binding, although high-affinity SNM1A binding requires the entire CSB C-terminal region (residues 1187-1493), where two copies of the C-terminal domain of CSB are necessary for a stable interaction with SNM1A. CSB stimulates SNM1A nuclease activity on varied model DNA repair intermediate substrates. Importantly, CSB was observed to stimulate digestion through ICLs in vitro , implying a key role of the interaction in 'unhooking' during TC-ICL repair. AlphaFold3 models of CSB constructs complexed with the SNM1A catalytic domain enabled mapping of the molecular contacts required for the CSB-SNM1A interaction. This identified specific protein-protein interactions necessary for CSB's stimulation of SNM1A's activity that we confirmed experimentally. Additionally, our studies reveal the C-terminal region of CSB as a novel DNA binding region that also is involved in stimulation of SNM1A-mediated ICL repair. Moreover, targeting protein-protein interactions that are vital for specific nuclease activities, such as CSB's stimulation of SNM1A's nuclease activity, may be a productive alternative therapeutic strategy to nuclease active site inhibition.

19.
Nat Commun ; 15(1): 5392, 2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-38918391

RESUMO

DNA double-strand breaks (DSBs), such as those produced by radiation and radiomimetics, are amongst the most toxic forms of cellular damage, in part because they involve extensive oxidative modifications at the break termini. Prior to completion of DSB repair, the chemically modified termini must be removed. Various DNA processing enzymes have been implicated in the processing of these dirty ends, but molecular knowledge of this process is limited. Here, we demonstrate a role for the metallo-ß-lactamase fold 5'-3' exonuclease SNM1A in this vital process. Cells disrupted for SNM1A manifest increased sensitivity to radiation and radiomimetic agents and show defects in DSB damage repair. SNM1A is recruited and is retained at the sites of DSB damage via the concerted action of its three highly conserved PBZ, PIP box and UBZ interaction domains, which mediate interactions with poly-ADP-ribose chains, PCNA and the ubiquitinated form of PCNA, respectively. SNM1A can resect DNA containing oxidative lesions induced by radiation damage at break termini. The combined results reveal a crucial role for SNM1A to digest chemically modified DNA during the repair of DSBs and imply that the catalytic domain of SNM1A is an attractive target for potentiation of radiotherapy.


Assuntos
Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA , Reparo do DNA , Exodesoxirribonucleases , Humanos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Exodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/genética , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , DNA/metabolismo , DNA/genética , Ubiquitinação , Proteínas de Ciclo Celular
20.
Chem Sci ; 15(21): 8227-8241, 2024 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-38817593

RESUMO

The three human SNM1 metallo-ß-lactamase fold nucleases (SNM1A-C) play key roles in DNA damage repair and in maintaining telomere integrity. Genetic studies indicate that they are attractive targets for cancer treatment and to potentiate chemo- and radiation-therapy. A high-throughput screen for SNM1A inhibitors identified diverse pharmacophores, some of which were shown by crystallography to coordinate to the di-metal ion centre at the SNM1A active site. Structure and turnover assay-guided optimization enabled the identification of potent quinazoline-hydroxamic acid containing inhibitors, which bind in a manner where the hydroxamic acid displaces the hydrolytic water and the quinazoline ring occupies a substrate nucleobase binding site. Cellular assays reveal that SNM1A inhibitors cause sensitisation to, and defects in the resolution of, cisplatin-induced DNA damage, validating the tractability of MBL fold nucleases as cancer drug targets.

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