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1.
Nat Commun ; 11(1): 4784, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963245

RESUMO

Genomic integrity is threatened by cytotoxic DNA double-strand breaks (DSBs), which must be resolved efficiently to prevent sequence loss, chromosomal rearrangements/translocations, or cell death. Polymerase µ (Polµ) participates in DSB repair via the nonhomologous end-joining (NHEJ) pathway, by filling small sequence gaps in broken ends to create substrates ultimately ligatable by DNA Ligase IV. Here we present structures of human Polµ engaging a DSB substrate. Synapsis is mediated solely by Polµ, facilitated by single-nucleotide homology at the break site, wherein both ends of the discontinuous template strand are stabilized by a hydrogen bonding network. The active site in the quaternary Pol µ complex is poised for catalysis and nucleotide incoporation proceeds in crystallo. These structures demonstrate that Polµ may address complementary DSB substrates during NHEJ in a manner indistinguishable from single-strand breaks.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Polimerase Dirigida por DNA/química , DNA/química , Cristalografia por Raios X , Dano ao DNA , Reparo do DNA por Junção de Extremidades , DNA Ligase Dependente de ATP/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/química , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Conformação Proteica
2.
Nat Commun ; 11(1): 4196, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826907

RESUMO

Cells utilise specialized polymerases from the Primase-Polymerase (Prim-Pol) superfamily to maintain genome stability. Prim-Pol's function in genome maintenance pathways including replication, repair and damage tolerance. Mycobacteria contain multiple Prim-Pols required for lesion repair, including Prim-PolC that performs short gap repair synthesis during excision repair. To understand the molecular basis of Prim-PolC's gap recognition and synthesis activities, we elucidated crystal structures of pre- and post-catalytic complexes bound to gapped DNA substrates. These intermediates explain its binding preference for short gaps and reveal a distinctive modus operandi called Synthesis-dependent Template Displacement (STD). This mechanism enables Prim-PolC to couple primer extension with template base dislocation, ensuring that the unpaired templating bases in the gap are ushered into the active site in an ordered manner. Insights provided by these structures establishes the molecular basis of Prim-PolC's gap recognition and extension activities, while also illuminating the mechanisms of primer extension utilised by closely related Prim-Pols.


Assuntos
Proteínas de Bactérias/química , DNA Primase/química , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/química , DNA/química , Mycobacterium/genética , Mycobacterium/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , DNA/metabolismo , DNA Primase/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Modelos Moleculares , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas
3.
Nucleic Acids Res ; 48(15): 8545-8561, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32735661

RESUMO

A crucial bacterial strategy to avoid killing by antibiotics is to enter a growth arrested state, yet the molecular mechanisms behind this process remain elusive. The conditional overexpression of mazF, the endoribonuclease toxin of the MazEF toxin-antitoxin system in Staphylococcus aureus, is one approach to induce bacterial growth arrest, but its targets remain largely unknown. We used overexpression of mazF and high-throughput sequence analysis following the exact mapping of non-phosphorylated transcriptome ends (nEMOTE) technique to reveal in vivo toxin cleavage sites on a global scale. We obtained a catalogue of MazF cleavage sites and unearthed an extended MazF cleavage specificity that goes beyond the previously reported one. We correlated transcript cleavage and abundance in a global transcriptomic profiling during mazF overexpression. We observed that MazF affects RNA molecules involved in ribosome biogenesis, cell wall synthesis, cell division and RNA turnover and thus deliver a plausible explanation for how mazF overexpression induces stasis. We hypothesize that autoregulation of MazF occurs by directly modulating the MazEF operon, such as the rsbUVW genes that regulate the sigma factor SigB, including an observed cleavage site on the MazF mRNA that would ultimately play a role in entry and exit from bacterial stasis.


Assuntos
Proteínas de Ligação a DNA/genética , Endorribonucleases/genética , Proteínas de Escherichia coli/genética , Staphylococcus aureus/genética , Sistemas Toxina-Antitoxina/genética , Antibacterianos/farmacologia , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/química , Escherichia coli/genética , Humanos , Óperon/genética , RNA Mensageiro/genética , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidade , Especificidade por Substrato , Transcriptoma/genética
4.
Nucleic Acids Res ; 48(15): 8529-8544, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32738045

RESUMO

Myocyte enhancer factor-2B (MEF2B) has the unique capability of binding to its DNA target sites with a degenerate motif, while still functioning as a gene-specific transcriptional regulator. Identifying its DNA targets is crucial given regulatory roles exerted by members of the MEF2 family and MEF2B's involvement in B-cell lymphoma. Analyzing structural data and SELEX-seq experimental results, we deduced the DNA sequence and shape determinants of MEF2B target sites on a high-throughput basis in vitro for wild-type and mutant proteins. Quantitative modeling of MEF2B binding affinities and computational simulations exposed the DNA readout mechanisms of MEF2B. The resulting binding signature of MEF2B revealed distinct intricacies of DNA recognition compared to other transcription factors. MEF2B uses base readout at its half-sites combined with shape readout at the center of its degenerate motif, where A-tract polarity dictates nuances of binding. The predominant role of shape readout at the center of the core motif, with most contacts formed in the minor groove, differs from previously observed protein-DNA readout modes. MEF2B, therefore, represents a unique protein for studies of the role of DNA shape in achieving binding specificity. MEF2B-DNA recognition mechanisms are likely representative for other members of the MEF2 family.


Assuntos
Proteínas de Ligação a DNA/ultraestrutura , DNA/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Sequência de Aminoácidos/genética , Sítios de Ligação/genética , DNA/genética , Proteínas de Ligação a DNA/química , Humanos , Linfoma de Células B/genética , Linfoma de Células B/patologia , Proteínas de Domínio MADS/genética , Proteínas de Domínio MADS/ultraestrutura , Fatores de Transcrição MEF2/química , Fatores de Transcrição MEF2/ultraestrutura , Complexos Multiproteicos/genética , Conformação de Ácido Nucleico , Motivos de Nucleotídeos/genética , Ligação Proteica/genética
5.
Mol Cell ; 80(1): 102-113.e6, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32853547

RESUMO

Repair of covalent DNA-protein crosslinks (DPCs) by DNA-dependent proteases has emerged as an essential genome maintenance mechanism required for cellular viability and tumor suppression. However, how proteolysis is restricted to the crosslinked protein while leaving surrounding chromatin proteins unharmed has remained unknown. Using defined DPC model substrates, we show that the DPC protease SPRTN displays strict DNA structure-specific activity. Strikingly, SPRTN cleaves DPCs at or in direct proximity to disruptions within double-stranded DNA. In contrast, proteins crosslinked to intact double- or single-stranded DNA are not cleaved by SPRTN. NMR spectroscopy data suggest that specificity is not merely affinity-driven but achieved through a flexible bipartite strategy based on two DNA binding interfaces recognizing distinct structural features. This couples DNA context to activation of the enzyme, tightly confining SPRTN's action to biologically relevant scenarios.


Assuntos
Reagentes para Ligações Cruzadas/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/química , Linhagem Celular , Proteínas de Ligação a DNA/química , Humanos , Espectroscopia de Ressonância Magnética , Modelos Biológicos , Domínios Proteicos , Relação Estrutura-Atividade
6.
Nat Commun ; 11(1): 3398, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636384

RESUMO

SWI/SNF remodelers play a key role in regulating chromatin architecture and gene expression. Here, we report the cryo-EM structure of the Saccharomyces cerevisiae Swi/Snf complex in a nucleosome-free state. The structure consists of a stable triangular base module and a flexible Arp module. The conserved subunits Swi1 and Swi3 form the backbone of the complex and closely interact with other components. Snf6, which is specific for yeast Swi/Snf complex, stabilizes the binding of the ATPase-containing subunit Snf2 to the base module. Comparison of the yeast Swi/Snf and RSC complexes reveals conserved structural features that govern the assembly and function of these two subfamilies of chromatin remodelers. Our findings complement those from recent structures of the yeast and human chromatin remodelers and provide further insights into the assembly and function of the SWI/SNF remodelers.


Assuntos
Adenosina Trifosfatases/química , Cromatina/química , Proteínas Cromossômicas não Histona/química , Proteínas Nucleares/química , Proteínas Repressoras/química , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/química , Microscopia Crioeletrônica , Proteínas de Ligação a DNA/química , Humanos , Nucleossomos , Ligação Proteica , Domínios Proteicos , Saccharomyces cerevisiae/química
7.
Nat Commun ; 11(1): 3723, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709850

RESUMO

DNA methylation maintenance by DNMT1 is an essential process in mammals but molecular mechanisms connecting DNA methylation patterns and enzyme activity remain elusive. Here, we systematically analyzed the specificity of DNMT1, revealing a pronounced influence of the DNA sequences flanking the target CpG site on DNMT1 activity. We determined DNMT1 structures in complex with preferred DNA substrates revealing that DNMT1 employs flanking sequence-dependent base flipping mechanisms, with large structural rearrangements of the DNA correlating with low catalytic activity. Moreover, flanking sequences influence the conformational dynamics of the active site and cofactor binding pocket. Importantly, we show that the flanking sequence preferences of DNMT1 highly correlate with genomic methylation in human and mouse cells, and 5-azacytidine triggered DNA demethylation is more pronounced at CpG sites with flanks disfavored by DNMT1. Overall, our findings uncover the intricate interplay between CpG-flanking sequence, DNMT1-mediated base flipping and the dynamic landscape of DNA methylation.


Assuntos
Sequência de Bases , DNA (Citosina-5-)-Metiltransferase 1/química , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA , DNA/química , DNA/metabolismo , Animais , Domínio Catalítico , Cristalografia por Raios X , DNA (Citosina-5-)-Metiltransferase 1/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Técnicas de Inativação de Genes , Cinética , Camundongos Knockout , Modelos Moleculares , Conformação de Ácido Nucleico , Oligonucleotídeos , Conformação Proteica , Especificidade por Substrato
8.
Nat Commun ; 11(1): 3703, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32710080

RESUMO

Mycobacterium tuberculosis is a pathogen with a unique cell envelope including very long fatty acids, implicated in bacterial resistance and host immune modulation. FasR is a TetR-like transcriptional activator that plays a central role in sensing mycobacterial long-chain fatty acids and regulating lipid biosynthesis. Here we disclose crystal structures of M. tuberculosis FasR in complex with acyl effector ligands and with DNA, uncovering its molecular sensory and switching mechanisms. A long tunnel traverses the entire effector-binding domain, enabling long fatty acyl effectors to bind. Only when the tunnel is entirely occupied, the protein dimer adopts a rigid configuration with its DNA-binding domains in an open state, leading to DNA dissociation. The protein-folding hydrophobic core connects the two domains, and is completed into a continuous spine when the effector binds. Such a transmission spine is conserved in a large number of TetR-like regulators, offering insight into effector-triggered allosteric functional control.


Assuntos
Acil Coenzima A/química , Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Mycobacterium tuberculosis/metabolismo , Fatores de Transcrição/química , Acil Coenzima A/metabolismo , Sítio Alostérico , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Cristalografia por Raios X , DNA Bacteriano/química , Proteínas de Ligação a DNA/metabolismo , Ácidos Graxos/metabolismo , Ligantes , Modelos Moleculares , Conformação Proteica , Fatores de Transcrição/metabolismo
9.
BMC Bioinformatics ; 21(1): 322, 2020 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-32689927

RESUMO

BACKGROUND: The study of DNA binding protein (DBP)-drug interactions can open a breakthrough for the treatment of genetic diseases and cancers. Currently, network-based methods are widely used for protein-drug interaction prediction, and many hidden relationships can be found through network analysis. We proposed a DCA (drug-cluster association) model for predicting DBP-drug interactions. The clusters are some similarities in the drug-binding site trimmers with their physicochemical properties. First, DBPs-drug binding sites are extracted from scPDB database. Second, each binding site is represented as a trimer which is obtained by sliding the window in the binding sites. Third, the trimers are clustered based on the physicochemical properties. Fourth, we build the network by generating the interaction matrix for representing the DCA network. Fifth, three link prediction methods are detected in the network. Finally, the common neighbor (CN) method is selected to predict drug-cluster associations in the DBP-drug network model. RESULT: This network shows that drugs tend to bind to positively charged sites and the binding process is more likely to occur inside the DBPs. The results of the link prediction indicate that the CN method has better prediction performance than the PA and JA methods. The DBP-drug network prediction model is generated by using the CN method which predicted more accurately drug-trimer interactions and DBP-drug interactions. Such as, we found that Erythromycin (ERY) can establish an interaction relationship with HTH-type transcriptional repressor, which is fitted well with silico DBP-drug prediction. CONCLUSION: The drug and protein bindings are local events. The binding of the drug-DBPs binding site represents this local binding event, which helps to understand the mechanism of DBP-drug interactions.


Assuntos
Biologia Computacional/métodos , Proteínas de Ligação a DNA/metabolismo , Bases de Dados Factuais , Interações Medicamentosas , Preparações Farmacêuticas/metabolismo , Sítios de Ligação , Simulação por Computador , Proteínas de Ligação a DNA/química , Humanos , Preparações Farmacêuticas/química , Ligação Proteica
10.
Proc Natl Acad Sci U S A ; 117(31): 18858-18868, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32694206

RESUMO

Buried seedlings undergo dramatic developmental transitions when they emerge from soil into sunlight. As central transcription factors suppressing light responses, PHYTOCHROME-INTERACTING FACTORs (PIFs) and ETHYLENE-INSENSITIVE 3 (EIN3) actively function in darkness and must be promptly repressed upon light to initiate deetiolation. Microproteins are evolutionarily conserved small single-domain proteins that act as posttranslational regulators in eukaryotes. Although hundreds to thousands of microproteins are predicted to exist in plants, their target molecules, biological roles, and mechanisms of action remain largely unknown. Here, we show that two microproteins, miP1a and miP1b (miP1a/b), are robustly stimulated in the dark-to-light transition. miP1a/b are primarily expressed in cotyledons and hypocotyl, exhibiting tissue-specific patterns similar to those of PIFs and EIN3 We demonstrate that PIFs and EIN3 assemble functional oligomers by self-interaction, while miP1a/b directly interact with and disrupt the oligomerization of PIFs and EIN3 by forming nonfunctional protein complexes. As a result, the DNA binding capacity and transcriptional activity of PIFs and EIN3 are predominantly suppressed. These biochemical findings are further supported by genetic evidence. miP1a/b positively regulate photomorphogenic development, and constitutively expressing miP1a/b rescues the delayed apical hook unfolding and cotyledon development of plants overexpressing PIFs and EIN3 Our study reveals that microproteins provide a temporal and negative control of the master transcription factors' oligomerization to achieve timely developmental transitions upon environmental changes.


Assuntos
Proteínas de Arabidopsis , Proteínas de Ligação a DNA , Desenvolvimento Vegetal/efeitos da radiação , Transdução de Sinais/efeitos da radiação , Fatores de Transcrição , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , Especificidade de Órgãos , Multimerização Proteica/efeitos da radiação , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
11.
Nucleic Acids Res ; 48(15): 8755-8766, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32621606

RESUMO

The sulfur atom of phosphorothioated DNA (PT-DNA) is coordinated by a surface cavity in the conserved sulfur-binding domain (SBD) of type IV restriction enzymes. However, some SBDs cannot recognize the sulfur atom in some sequence contexts. To illustrate the structural determinants for sequence specificity, we resolved the structure of SBDSpr, from endonuclease SprMcrA, in complex with DNA of GPSGCC, GPSATC and GPSAAC contexts. Structural and computational analyses explained why it binds the above PT-DNAs with an affinity in a decreasing order. The structural analysis of SBDSpr-GPSGCC and SBDSco-GPSGCC, the latter only recognizes DNA of GPSGCC, revealed that a positively charged loop above the sulfur-coordination cavity electrostatically interacts with the neighboring DNA phosphate linkage. The structural analysis indicated that the DNA-protein hydrogen bonding pattern and weak non-bonded interaction played important roles in sequence specificity of SBD protein. Exchanges of the positively-charged amino acid residues with the negatively-charged residues in the loop would enable SBDSco to extend recognization for more PT-DNA sequences, implying that type IV endonucleases can be engineered to recognize PT-DNA in novel target sequences.


Assuntos
Enzimas de Restrição do DNA/genética , Proteínas de Ligação a DNA/genética , DNA/genética , Enxofre/química , Sequência de Aminoácidos/genética , Cristalografia por Raios X , DNA/química , Enzimas de Restrição do DNA/química , Proteínas de Ligação a DNA/química , Escherichia coli/genética , Ligação de Hidrogênio , Ligação Proteica/genética , Domínios Proteicos/genética , Streptomyces/enzimologia
12.
Proc Natl Acad Sci U S A ; 117(27): 15731-15739, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32561643

RESUMO

De novo emergence demands a transition from disordered polypeptides into structured proteins with well-defined functions. However, can polypeptides confer functions of evolutionary relevance, and how might such polypeptides evolve into modern proteins? The earliest proteins present an even greater challenge, as they were likely based on abiotic, spontaneously synthesized amino acids. Here we asked whether a primordial function, such as nucleic acid binding, could emerge with ornithine, a basic amino acid that forms abiotically yet is absent in modern-day proteins. We combined ancestral sequence reconstruction and empiric deconstruction to unravel a gradual evolutionary trajectory leading from a polypeptide to a ubiquitous nucleic acid-binding protein. Intermediates along this trajectory comprise sequence-duplicated functional proteins built from 10 amino acid types, with ornithine as the only basic amino acid. Ornithine side chains were further modified into arginine by an abiotic chemical reaction, improving both structure and function. Along this trajectory, function evolved from phase separation with RNA (coacervates) to avid and specific double-stranded DNA binding. Our results suggest that phase-separating polypeptides may have been an evolutionary resource for the emergence of early proteins, and that ornithine, together with its postsynthesis modification to arginine, could have been the earliest basic amino acids.


Assuntos
Arginina/química , Nucleoproteínas/genética , Ornitina/química , Peptídeos/genética , Sequência de Aminoácidos/genética , Aminoácidos/química , Aminoácidos/genética , Arginina/genética , DNA/química , DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Nucleoproteínas/química , Ornitina/genética , Peptídeos/química , Proteínas/química , Proteínas/genética , RNA/química , RNA/genética
13.
Proc Natl Acad Sci U S A ; 117(28): 16302-16312, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32586954

RESUMO

DNA mismatch repair (MMR) corrects errors that occur during DNA replication. In humans, mutations in the proteins MutSα and MutLα that initiate MMR cause Lynch syndrome, the most common hereditary cancer. MutSα surveilles the DNA, and upon recognition of a replication error it undergoes adenosine triphosphate-dependent conformational changes and recruits MutLα. Subsequently, proliferating cell nuclear antigen (PCNA) activates MutLα to nick the error-containing strand to allow excision and resynthesis. The structure-function properties of these obligate MutSα-MutLα complexes remain mostly unexplored in higher eukaryotes, and models are predominately based on studies of prokaryotic proteins. Here, we utilize atomic force microscopy (AFM) coupled with other methods to reveal time- and concentration-dependent stoichiometries and conformations of assembling human MutSα-MutLα-DNA complexes. We find that they assemble into multimeric complexes comprising three to eight proteins around a mismatch on DNA. On the timescale of a few minutes, these complexes rearrange, folding and compacting the DNA. These observations contrast with dominant models of MMR initiation that envision diffusive MutS-MutL complexes that move away from the mismatch. Our results suggest MutSα localizes MutLα near the mismatch and promotes DNA configurations that could enhance MMR efficiency by facilitating MutLα nicking the DNA at multiple sites around the mismatch. In addition, such complexes may also protect the mismatch region from nucleosome reassembly until repair occurs, and they could potentially remodel adjacent nucleosomes.


Assuntos
Reparo de Erro de Pareamento de DNA , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas MutL/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Trifosfato de Adenosina/metabolismo , DNA/química , DNA/genética , Proteínas de Ligação a DNA/química , Humanos , Complexos Multiproteicos/metabolismo , Proteínas MutL/química , Proteína 2 Homóloga a MutS/química , Conformação de Ácido Nucleico , Nucleossomos/metabolismo , Dobramento de Proteína , Multimerização Proteica
14.
Nat Commun ; 11(1): 2728, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483114

RESUMO

The Pseudomonas putida phenol-responsive regulator DmpR is a bacterial enhancer binding protein (bEBP) from the AAA+ ATPase family. Even though it was discovered more than two decades ago and has been widely used for aromatic hydrocarbon sensing, the activation mechanism of DmpR has remained elusive. Here, we show that phenol-bound DmpR forms a tetramer composed of two head-to-head dimers in a head-to-tail arrangement. The DmpR-phenol complex exhibits altered conformations within the C-termini of the sensory domains and shows an asymmetric orientation and angle in its coiled-coil linkers. The structural changes within the phenol binding sites and the downstream ATPase domains suggest that the effector binding signal is propagated through the coiled-coil helixes. The tetrameric DmpR-phenol complex interacts with the σ54 subunit of RNA polymerase in presence of an ATP analogue, indicating that DmpR-like bEBPs tetramers utilize a mechanistic mode distinct from that of hexameric AAA+ ATPases to activate σ54-dependent transcription.


Assuntos
Adenosina Trifosfatases/química , Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Conformação Proteica , Multimerização Proteica , Transativadores/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Fenol/metabolismo , Ligação Proteica , Pseudomonas putida/enzimologia , Pseudomonas putida/genética , Homologia de Sequência de Aminoácidos , Transativadores/genética , Transativadores/metabolismo
15.
Proc Natl Acad Sci U S A ; 117(25): 14127-14138, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32522879

RESUMO

Xeroderma pigmentosum group G (XPG) protein is both a functional partner in multiple DNA damage responses (DDR) and a pathway coordinator and structure-specific endonuclease in nucleotide excision repair (NER). Different mutations in the XPG gene ERCC5 lead to either of two distinct human diseases: Cancer-prone xeroderma pigmentosum (XP-G) or the fatal neurodevelopmental disorder Cockayne syndrome (XP-G/CS). To address the enigmatic structural mechanism for these differing disease phenotypes and for XPG's role in multiple DDRs, here we determined the crystal structure of human XPG catalytic domain (XPGcat), revealing XPG-specific features for its activities and regulation. Furthermore, XPG DNA binding elements conserved with FEN1 superfamily members enable insights on DNA interactions. Notably, all but one of the known pathogenic point mutations map to XPGcat, and both XP-G and XP-G/CS mutations destabilize XPG and reduce its cellular protein levels. Mapping the distinct mutation classes provides structure-based predictions for disease phenotypes: Residues mutated in XP-G are positioned to reduce local stability and NER activity, whereas residues mutated in XP-G/CS have implied long-range structural defects that would likely disrupt stability of the whole protein, and thus interfere with its functional interactions. Combined data from crystallography, biochemistry, small angle X-ray scattering, and electron microscopy unveil an XPG homodimer that binds, unstacks, and sculpts duplex DNA at internal unpaired regions (bubbles) into strongly bent structures, and suggest how XPG complexes may bind both NER bubble junctions and replication forks. Collective results support XPG scaffolding and DNA sculpting functions in multiple DDR processes to maintain genome stability.


Assuntos
Síndrome de Cockayne/genética , Proteínas de Ligação a DNA/química , Endonucleases/química , Proteínas Nucleares/química , Mutação Puntual , Fatores de Transcrição/química , Xeroderma Pigmentoso/genética , Sítios de Ligação , Sequência Conservada , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Estabilidade Enzimática , Humanos , Simulação de Dinâmica Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Ligação Proteica , Dobramento de Proteína , Multimerização Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
16.
Mol Cell ; 79(3): 459-471.e4, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32553192

RESUMO

Transcription factors (TFs) that bind common DNA motifs in vitro occupy distinct sets of promoters in vivo, raising the question of how binding specificity is achieved. TFs are enriched with intrinsically disordered regions (IDRs). Such regions commonly form promiscuous interactions, yet their unique properties might also benefit specific binding-site selection. We examine this using Msn2 and Yap1, TFs of distinct families that contain long IDRs outside their DNA-binding domains. We find that these IDRs are both necessary and sufficient for localizing to the majority of target promoters. This IDR-directed binding does not depend on any localized domain but results from a multitude of weak determinants distributed throughout the entire IDR sequence. Furthermore, IDR specificity is conserved between distant orthologs, suggesting direct interaction with multiple promoters. We propose that distribution of sensing determinants along extended IDRs accelerates binding-site detection by rapidly localizing TFs to broad DNA regions surrounding these sites.


Assuntos
Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Proteínas Intrinsicamente Desordenadas/genética , Motivos de Nucleotídeos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Deleção de Sequência , Fatores de Transcrição/genética , Sítios de Ligação , Biologia Computacional/métodos , Sequência Conservada , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Estatísticos , Regiões Promotoras Genéticas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
17.
Nucleic Acids Res ; 48(13): 7421-7438, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32496517

RESUMO

The long non-coding RNA NEAT1 serves as a scaffold for the assembly of paraspeckles, membraneless nuclear organelles involved in gene regulation. Paraspeckle assembly requires NEAT1 recruitment of the RNA-binding protein NONO, however the NEAT1 elements responsible for recruitment are unknown. Herein we present evidence that previously unrecognized structural features of NEAT1 serve an important role in these interactions. Led by the initial observation that NONO preferentially binds the G-quadruplex conformation of G-rich C9orf72 repeat RNA, we find that G-quadruplex motifs are abundant and conserved features of NEAT1. Furthermore, we determine that NONO binds NEAT1 G-quadruplexes with structural specificity and provide evidence that G-quadruplex motifs mediate NONO-NEAT1 association, with NONO binding sites on NEAT1 corresponding largely to G-quadruplex motifs, and treatment with a G-quadruplex-disrupting small molecule causing dissociation of native NONO-NEAT1 complexes. Together, these findings position G-quadruplexes as a primary candidate for the NONO-recruiting elements of NEAT1 and provide a framework for further investigation into the role of G-quadruplexes in paraspeckle formation and function.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Quadruplex G , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Sítios de Ligação , Sequência Conservada , Proteínas de Ligação a DNA/química , Células HEK293 , Humanos , Camundongos , Ligação Proteica , RNA Longo não Codificante/química , Proteínas de Ligação a RNA/química
18.
PLoS Genet ; 16(5): e1008797, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32392219

RESUMO

Sun-loving plants perceive the proximity of potential light-competing neighboring plants as a reduction in the red:far-red ratio (R:FR), which elicits a suite of responses called the "shade avoidance syndrome" (SAS). Changes in R:FR are primarily perceived by phytochrome B (phyB), whereas UV-B perceived by UV RESISTANCE LOCUS 8 (UVR8) elicits opposing responses to provide a counterbalance to SAS, including reduced shade-induced hypocotyl and petiole elongation. Here we show at the genome-wide level that UVR8 broadly suppresses shade-induced gene expression. A subset of this gene regulation is dependent on the UVR8-stabilized atypical bHLH transcription regulator LONG HYPOCOTYL IN FAR-RED 1 (HFR1), which functions in part redundantly with PHYTOCHROME INTERACTING FACTOR 3-LIKE 1 (PIL1). In parallel, UVR8 signaling decreases protein levels of the key positive regulators of SAS, namely the bHLH transcription factors PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and PIF5, in a COP1-dependent but HFR1-independent manner. We propose that UV-B antagonizes SAS via two mechanisms: degradation of PIF4 and PIF5, and HFR1- and PIL1-mediated inhibition of PIF4 and PIF5 function. This work highlights the importance of typical and atypical bHLH transcription regulators for the integration of light signals from different photoreceptors and provides further mechanistic insight into the crosstalk of UVR8 signaling and SAS.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/química , Raios Ultravioleta/efeitos adversos , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Estabilidade Proteica , Proteólise , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
19.
Nucleic Acids Res ; 48(10): 5235-5253, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32356888

RESUMO

Antisense oligonucleotides (ASOs) interact with target RNAs via hybridization to modulate gene expression through different mechanisms. ASO therapeutics are chemically modified and include phosphorothioate (PS) backbone modifications and different ribose and base modifications to improve pharmacological properties. Modified PS ASOs display better binding affinity to the target RNAs and increased binding to proteins. Moreover, PS ASO protein interactions can affect many aspects of their performance, including distribution and tissue delivery, cellular uptake, intracellular trafficking, potency and toxicity. In this review, we summarize recent progress in understanding PS ASO protein interactions, highlighting the proteins with which PS ASOs interact, the influence of PS ASO protein interactions on ASO performance, and the structure activity relationships of PS ASO modification and protein interactions. A detailed understanding of these interactions can aid in the design of safer and more potent ASO drugs, as illustrated by recent findings that altering ASO chemical modifications dramatically improves therapeutic index.


Assuntos
Oligonucleotídeos Fosforotioatos/química , Proteínas/química , Membrana Celular/química , Membrana Celular/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Espaço Intracelular/química , Espaço Intracelular/metabolismo , Ligantes , Oligonucleotídeos Fosforotioatos/metabolismo , Oligonucleotídeos Fosforotioatos/farmacologia , Oligonucleotídeos Fosforotioatos/toxicidade , Ligação Proteica , Domínios Proteicos , Proteínas/metabolismo , Proteínas/toxicidade , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribonuclease H/química , Ribonuclease H/metabolismo , Relação Estrutura-Atividade , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
20.
PLoS Comput Biol ; 16(5): e1007867, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32453726

RESUMO

DNA sequences are often recognized by multi-domain proteins that may have higher affinity and specificity than single-domain proteins. However, the higher affinity to DNA might be coupled with slower recognition kinetics. In this study, we address this balance between stability and kinetics for multi-domain Cys2His2- (C2H2-) type zinc-finger (ZF) proteins. These proteins are the most prevalent DNA-binding domain in eukaryotes and C2H2 type zinc-finger proteins (C2H2-ZFPs) constitute nearly one-half of all known and predicted transcription factors in human. Extensive contact with DNA via tandem ZF domains confers high stability on the sequence-specific complexes. However, this can limit target search efficiency, especially for low abundance ZFPs. Earlier, we found that asymmetrical distribution of electrostatic charge among the three ZF domains of the low abundance transcription factor Egr-1 facilitates its DNA search process. Here, on a diverse set of 273 human C2H2-ZFP comprised of 3-15 tandem ZF domains, we find that, in many cases, electrostatic charge and binding specificity are asymmetrically distributed among the ZF domains so that neighbouring domains have different DNA-binding properties. For proteins containing 3-6 ZF domains, we show that the low abundance proteins possess a higher degree of non-specific asymmetry and vice versa. Our findings suggest that where the electrostatics of tandem ZF domains are similar (i.e., symmetrical), the ZFPs are more abundant to optimize their DNA search efficiency. This study reveals new insights into the fundamental determinants of recognition by C2H2-ZFPs of their DNA binding sites in the cellular landscape. The importance of electrostatic asymmetry with respect to binding site recognition by C2H2-ZFPs suggests the possibility that it may also be important in other ZFP systems and reveals a new design feature for zinc finger engineering.


Assuntos
Proteínas de Ligação a DNA/química , DNA/química , Regulação da Expressão Gênica , Dedos de Zinco , Sítios de Ligação , Proteína 1 de Resposta de Crescimento Precoce/química , Humanos , Cinética , Ligação Proteica , Domínios Proteicos , Eletricidade Estática , Fator de Transcrição YY1/química
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