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1.
J Phys Chem Lett ; 12(32): 7878-7884, 2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34382809

RESUMO

The histone-like nucleoid structuring (H-NS) protein controls the expression of hundreds of genes in Gram-positive bacteria through its capability to coat and condense DNA. This mechanism requires the formation of superhelical H-NS protein filaments that are sensitive to temperature and salinity, allowing H-NS to act as an environment sensor. We use multiscale modeling and simulations to obtain detailed insights into the mechanism of H-NS filament's sensitivity to environmental changes. Through the simulations of the superhelical H-NS filament, we reveal how different environments induce heterogeneity of H-NS monomers. Further, we observe that transient self-association within the H-NS filament creates temperature-inducible strain and might mildly oppose DNA binding. We also probe different H-NS-DNA complex architectures and show that complexation enhances the stability of both DNA and H-NS superhelices. Overall, our results provide unprecedented molecular insights into the environmental sensing and DNA interactions of a prototypical nucleoid-structuring bacterial protein filament.


Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Salinidade , Salmonella typhimurium/química , Temperatura
2.
Science ; 373(6557): 882-889, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34413232

RESUMO

Eukaryotic genomes contain domesticated genes from integrating viruses and mobile genetic elements. Among these are homologs of the capsid protein (known as Gag) of long terminal repeat (LTR) retrotransposons and retroviruses. We identified several mammalian Gag homologs that form virus-like particles and one LTR retrotransposon homolog, PEG10, that preferentially binds and facilitates vesicular secretion of its own messenger RNA (mRNA). We showed that the mRNA cargo of PEG10 can be reprogrammed by flanking genes of interest with Peg10's untranslated regions. Taking advantage of this reprogrammability, we developed selective endogenous encapsidation for cellular delivery (SEND) by engineering both mouse and human PEG10 to package, secrete, and deliver specific RNAs. Together, these results demonstrate that SEND is a modular platform suited for development as an efficient therapeutic delivery modality.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação a DNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Capsídeo/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Vesículas Extracelulares/metabolismo , Edição de Genes , Vetores Genéticos , Humanos , Camundongos , Neurônios/metabolismo , Domínios Proteicos , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Retroelementos , Transfecção , Regiões não Traduzidas , Regulação para Cima
3.
Nat Commun ; 12(1): 4760, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362909

RESUMO

The MADS transcription factors (TF) are an ancient eukaryotic protein family. In plants, the family is divided into two main lineages. Here, we demonstrate that DNA binding in both lineages absolutely requires a short amino acid sequence C-terminal to the MADS domain (M domain) called the Intervening domain (I domain) that was previously defined only in type II lineage MADS. Structural elucidation of the MI domains from the floral regulator, SEPALLATA3 (SEP3), shows a conserved fold with the I domain acting to stabilise the M domain. Using the floral organ identity MADS TFs, SEP3, APETALA1 (AP1) and AGAMOUS (AG), domain swapping demonstrate that the I domain alters genome-wide DNA-binding specificity and dimerisation specificity. Introducing AG carrying the I domain of AP1 in the Arabidopsis ap1 mutant resulted in strong complementation and restoration of first and second whorl organs. Taken together, these data demonstrate that the I domain acts as an integral part of the DNA-binding domain and significantly contributes to the functional identity of the MADS TF.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Homeodomínio/química , Fatores de Transcrição/química , Proteína AGAMOUS de Arabidopsis/química , Proteína AGAMOUS de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Ligação a DNA/metabolismo , Flores , Regulação da Expressão Gênica de Plantas , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Domínio MADS/metabolismo , Fenótipo , Domínios e Motivos de Interação entre Proteínas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
Biomolecules ; 11(7)2021 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-34356632

RESUMO

Ubiquitin (Ub) specifically interacts with the Ub-associating domain (UBA) in a proteasomal shuttle factor, while the latter is involved in either proteasomal targeting or self-assembly coacervation. PINK1 phosphorylates Ub at S65 and makes Ub alternate between C-terminally relaxed (pUbRL) and retracted conformations (pUbRT). Using NMR spectroscopy, we show that pUbRL but not pUbRT preferentially interacts with the UBA from two proteasomal shuttle factors Ubqln2 and Rad23A. Yet discriminatorily, Ubqln2-UBA binds to pUb more tightly than Rad23A does and selectively enriches pUbRL upon complex formation. Further, we determine the solution structure of the complex between Ubqln2-UBA and pUbRL and uncover the thermodynamic basis for the stronger interaction. NMR kinetics analysis at different timescales further suggests an indued-fit binding mechanism for pUb-UBA interaction. Notably, at a relatively low saturation level, the dissociation rate of the UBA-pUbRL complex is comparable with the exchange rate between pUbRL and pUbRT. Thus, a kinetic constraint would dictate the interaction between Ub and UBA, thus fine-tuning the functional state of the proteasomal shuttle factors.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Relacionadas à Autofagia/química , Enzimas Reparadoras do DNA/química , Proteínas de Ligação a DNA/química , Proteínas Quinases/química , Ubiquitina/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Domínios Proteicos , Proteínas Quinases/metabolismo , Termodinâmica , Ubiquitina/metabolismo
5.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360978

RESUMO

Transactive response DNA-binding protein 43 (TDP-43) is a nucleic acid-binding protein that is involved in transcription and translation regulation, non-coding RNA processing, and stress granule assembly. Aside from its multiple functions, it is also known as the signature protein in the hallmark inclusions of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) patients. TDP-43 is built of four domains, but its low-complexity domain (LCD) has become an intense research focus that brings to light its possible role in TDP-43 functions and involvement in the pathogenesis of these neurodegenerative diseases. Recent endeavors have further uncovered the distinct biophysical properties of TDP-43 under various circumstances. In this review, we summarize the multiple structural and biochemical properties of LCD in either promoting the liquid droplets or inducing fibrillar aggregates. We also revisit the roles of the LCD in paraspeckles, stress granules, and cytoplasmic inclusions to date.


Assuntos
Amiloide/metabolismo , Proteínas de Ligação a DNA/química , Gotículas Lipídicas/metabolismo , Proteinopatias TDP-43/metabolismo , Animais , Grânulos Citoplasmáticos/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Domínios Proteicos
6.
Int J Mol Sci ; 22(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34360641

RESUMO

The cyclic AMP receptor protein (CRP) is one of the best-known transcription factors, regulating about 400 genes. The histone-like nucleoid structuring protein (H-NS) is one of the nucleoid-forming proteins and is responsible for DNA packaging and gene repression in prokaryotes. In this study, the binding of ppGpp to CRP and H-NS was determined by fluorescence spectroscopy. CRP from Escherichia coli exhibited intrinsic fluorescence at 341 nm when excited at 280 nm. The fluorescence intensity decreased in the presence of ppGpp. The dissociation constant of 35 ± 3 µM suggests that ppGpp binds to CRP with a similar affinity to cAMP. H-NS also shows intrinsic fluorescence at 329 nm. The fluorescence intensity was decreased by various ligands and the calculated dissociation constant for ppGpp was 80 ± 11 µM, which suggests that the binding site was occupied fully by ppGpp under starvation conditions. This study suggests the modulatory effects of ppGpp in gene expression regulated by CRP and H-NS. The method described here may be applicable to many other proteins.


Assuntos
Proteínas de Bactérias/metabolismo , Proteína Receptora de AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Guanosina Tetrafosfato/metabolismo , Proteínas de Bactérias/química , Sítios de Ligação , AMP Cíclico/metabolismo , Proteína Receptora de AMP Cíclico/química , Proteínas de Ligação a DNA/química , Escherichia coli , Espectrometria de Fluorescência
7.
Nat Commun ; 12(1): 4057, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34210977

RESUMO

Chromatin remodeler ALC1 (amplification in liver cancer 1) is crucial for repairing damaged DNA. It is autoinhibited and activated by nucleosomal epitopes. However, the mechanisms by which ALC1 is regulated remain unclear. Here we report the crystal structure of human ALC1 and the cryoEM structure bound to the nucleosome. The structure shows the macro domain of ALC1 binds to lobe 2 of the ATPase motor, sequestering two elements for nucleosome recognition, explaining the autoinhibition mechanism of the enzyme. The H4 tail competes with the macro domain for lobe 2-binding, explaining the requirement for this nucleosomal epitope for ALC1 activation. A dual-arginine-anchor motif of ALC1 recognizes the acidic pocket of the nucleosome, which is critical for chromatin remodeling in vitro. Together, our findings illustrate the structures of ALC1 and shed light on its regulation mechanisms, paving the way for the discovery of drugs targeting ALC1 for the treatment of cancer.


Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Cristalografia por Raios X/métodos , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/química , Neoplasias Hepáticas/patologia , Nucleossomos/metabolismo , Proteínas Recombinantes/química , Células Cultivadas , DNA Helicases/química , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Domínios Proteicos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade
8.
FEBS Lett ; 595(16): 2071-2084, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34245011

RESUMO

Human coilin-interacting nuclear ATPase protein (hCINAP), also known as adenylate kinase 6 (AK6), is an atypical adenylate kinase with critical roles in many biological processes, including gene transcription, ribosome synthesis, cell metabolism, cell proliferation and apoptosis, DNA damage responses, and genome stability. Furthermore, hCINAP/AK6 dysfunction is associated with cancer and various inflammatory diseases. In this review, we summarize the structural features and biological roles of hCINAP in several important signaling pathways, as well as its connection with tumor onset and progression.


Assuntos
Carcinogênese , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Progressão da Doença , Neoplasias/enzimologia , Neoplasias/patologia , Humanos
9.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205216

RESUMO

Two independent, complementary methods of structural analysis were used to elucidate the effect of divalent magnesium and iron cations on the structure of the protective Dps-DNA complex. Small-angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-EM) demonstrate that Mg2+ ions block the N-terminals of the Dps protein preventing its interaction with DNA. Non-interacting macromolecules of Dps and DNA remain in the solution in this case. The subsequent addition of the chelating agent (EDTA) leads to a complete restoration of the structure of the complex. Different effect was observed when Fe cations were added to the Dps-DNA complex; the presence of Fe2+ in solution leads to the total complex destruction and aggregation without possibility of the complex restoration with the chelating agent. Here, we discuss these different responses of the Dps-DNA complex on the presence of additional free metal cations, investigating the structure of the Dps protein with and without cations using SAXS and cryo-EM. Additionally, the single particle analysis of Dps with accumulated iron performed by cryo-EM shows localization of iron nanoparticles inside the Dps cavity next to the acidic (hydrophobic) pore, near three glutamate residues.


Assuntos
Proteínas da Membrana Bacteriana Externa/ultraestrutura , DNA/ultraestrutura , Proteínas de Escherichia coli/ultraestrutura , Ferro/química , Magnésio/química , Sequência de Aminoácidos/efeitos dos fármacos , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Cátions/química , Microscopia Crioeletrônica , DNA/química , DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/ultraestrutura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Espalhamento a Baixo Ângulo , Difração de Raios X
10.
Molecules ; 26(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34210101

RESUMO

Artemis is an endonuclease responsible for breaking hairpin DNA strands during immune system adaptation and maturation as well as the processing of potentially toxic DNA lesions. Thus, Artemis may be an important target in the development of anticancer therapy, both for the sensitization of radiotherapy and for immunotherapy. Despite its importance, its structure has been resolved only recently, and important questions concerning the arrangement of its active center, the interaction with the DNA substrate, and the catalytic mechanism remain unanswered. In this contribution, by performing extensive molecular dynamic simulations, both classically and at the hybrid quantum mechanics/molecular mechanics level, we evidenced the stable interaction modes of Artemis with a model DNA strand. We also analyzed the catalytic cycle providing the free energy profile and key transition states for the DNA cleavage reaction.


Assuntos
Proteínas de Ligação a DNA/química , DNA/química , Endonucleases/química , Modelos Químicos , Humanos
11.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202641

RESUMO

The cohesin complex is crucial for mediating sister chromatid cohesion and for hierarchal three-dimensional organization of the genome. Mutations in cohesin genes are present in a range of cancers. Extensive research over the last few years has shown that cohesin mutations are key events that contribute to neoplastic transformation. Cohesin is involved in a range of cellular processes; therefore, the impact of cohesin mutations in cancer is complex and can be cell context dependent. Candidate targets with therapeutic potential in cohesin mutant cells are emerging from functional studies. Here, we review emerging targets and pharmacological agents that have therapeutic potential in cohesin mutant cells.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Predisposição Genética para Doença , Mutação , Neoplasias/genética , Biomarcadores Tumorais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Gerenciamento Clínico , Regulação da Expressão Gênica , Humanos , Terapia de Alvo Molecular/métodos , Neoplasias/diagnóstico , Neoplasias/metabolismo , Neoplasias/terapia , Especificidade de Órgãos , Ligação Proteica , Relação Estrutura-Atividade
12.
Phys Chem Chem Phys ; 23(24): 13745-13751, 2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34159970

RESUMO

DNA damage leads to stalled or collapsed replication forks. Replication restart primosomes re-initiate DNA synthesis at these stalled or collapsed DNA replication forks, which is important for bacterial survival. Primosomal protein PriA specifically recognizes the DNA fork structure and recruits other primosomal proteins to load the replicative helicase, in order to re-establish the replication fork. PriA binding on DNA is the first step to restart replication forks for proper DNA repair. Using a single-molecule fluorescence colocalization experiment, we measured the thermodynamic and real-time kinetic properties of fluorescence-labeled Gram-positive bacteria Geobacillus stearothermophilus PriA binding on DNA forks. We showed that PriA preferentially binds to a DNA fork structure with a fully duplexed leading strand at sub-nanomolar affinity (Kd = 268 ± 99 pM). PriA binds dynamically, and its association and dissociation rate constants can be determined using the appearance and disappearance of the fluorescence signal. In addition, we showed that PriA binds to DNA forks as a monomer using photobleaching step counting. This information offers a molecular basis essential for understanding the mechanism of replication restart.


Assuntos
Proteínas de Bactérias/química , DNA Bacteriano/química , Proteínas de Ligação a DNA/química , Geobacillus stearothermophilus/química , Sítios de Ligação , Replicação do DNA , Imagem Óptica
13.
Nat Methods ; 18(7): 816-820, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34127856

RESUMO

Single-molecule Förster resonance energy transfer (smFRET) has become a versatile and widespread method to probe nanoscale conformation and dynamics. However, current experimental modalities often resort to molecule immobilization for long observation times and do not always approach the resolution limit of FRET-based nanoscale metrology. Here we present ABEL-FRET, an immobilization-free platform for smFRET measurements with ultrahigh resolving power in FRET efficiency. Importantly, single-molecule diffusivity is used to provide additional size and shape information for hydrodynamic profiling of individual molecules, which, together with the concurrently measured intramolecular conformation through FRET, enables a holistic and dynamic view of biomolecules and their complexes.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Dano ao DNA , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/química , Transferência Ressonante de Energia de Fluorescência/instrumentação , Hidrodinâmica , Dispositivos Lab-On-A-Chip , Conformação Molecular , Ácidos Nucleicos Heteroduplexes/química , Fótons , Imagem Individual de Molécula/instrumentação
14.
Nat Commun ; 12(1): 3338, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099686

RESUMO

The versatile nucleotide excision repair (NER) pathway initiates as the XPC-RAD23B-CETN2 complex first recognizes DNA lesions from the genomic DNA and recruits the general transcription factor complex, TFIIH, for subsequent lesion verification. Here, we present a cryo-EM structure of an NER initiation complex containing Rad4-Rad23-Rad33 (yeast homologue of XPC-RAD23B-CETN2) and 7-subunit coreTFIIH assembled on a carcinogen-DNA adduct lesion at 3.9-9.2 Å resolution. A ~30-bp DNA duplex could be mapped as it straddles between Rad4 and the Ssl2 (XPB) subunit of TFIIH on the 3' and 5' side of the lesion, respectively. The simultaneous binding with Rad4 and TFIIH was permitted by an unwinding of DNA at the lesion. Translocation coupled with torque generation by Ssl2 and Rad4 would extend the DNA unwinding at the lesion and deliver the damaged strand to Rad3 (XPD) in an open form suitable for subsequent lesion scanning and verification.


Assuntos
Microscopia Crioeletrônica , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/química , DNA/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Fator de Transcrição TFIIH/química , Adutos de DNA/metabolismo , DNA Helicases/química , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Fator de Transcrição TFIIH/genética
15.
Phytomedicine ; 88: 153598, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34111615

RESUMO

INTRODUCTION: Depression is one of the leading causes of death worldwide. Lower antioxidant concentrations and increased oxidative stress levels contribute to the development of depression. Effective and tolerable medications are urgently needed. Nrf2 and PRDX2 are promising targets in the treatment of oxidative stress and, therefore, promising for the development of novel antidepressants. Ursolic acid (UA), a natural triterpenoid found in various plants is known to exert neuroprotective and antioxidant effects. Skn-1 (which corresponds to human Nrf2) and prdx2 deficient mutants of the nematode Caenorhabditis elegans are suitable models to study the effect of UA on these targets. Additionally, stress assays are used to mimic stress or depressed state. METHODS: We examined the antioxidant activity of UA in Caenorhabditis elegans wildtype and skn-1- and prdx2-deficient strains by H2DCF-DA and juglone assays as well as osmotic and heat stress assays. Additionally, we analyzed the binding of UA to human PRDX2 and Skn-1 proteins by molecular docking and microscale thermophoresis. RESULTS: UA exerted strong antioxidant activities. Additionally, induction of stress resistance towards osmotic and heat stress was observed. qRT-PCR revealed that UA upregulated the gene expression of skn-1 and prdx2. Molecular docking studies supported these findings. CONCLUSION: Our findings implicate that the strong antioxidant activity of UA may exert anti-depressive effects by its interaction with the Skn-1 transcription factor, which is part of a detoxification network, and the antioxidant PRDX2 protein, which protects the organism from the detrimental effects of radical oxygen species.


Assuntos
Caenorhabditis elegans/efeitos dos fármacos , Depressão/genética , Estresse Fisiológico/efeitos dos fármacos , Triterpenos/farmacologia , Animais , Antidepressivos/farmacologia , Antioxidantes/farmacologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Simulação de Acoplamento Molecular , Mutação , Estresse Oxidativo/efeitos dos fármacos , Peroxirredoxinas/genética , Substâncias Protetoras/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Triterpenos/química
16.
Nucleic Acids Res ; 49(11): 6569-6586, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34107018

RESUMO

Replicative helicases are essential proteins that unwind DNA in front of replication forks. Their loading depends on accessory proteins and in bacteria, DnaC and DnaI are well characterized loaders. However, most bacteria do not express either of these two proteins. Instead, they are proposed to rely on DciA, an ancestral protein unrelated to DnaC/I. While the DciA structure from Vibrio cholerae shares no homology with DnaC, it reveals similarities with DnaA and DnaX, two proteins involved during replication initiation. As other bacterial replicative helicases, VcDnaB adopts a toroid-shaped homo-hexameric structure, but with a slightly open dynamic conformation in the free state. We show that VcDnaB can load itself on DNA in vitro and that VcDciA stimulates this function, resulting in an increased DNA unwinding. VcDciA interacts with VcDnaB with a 3/6 stoichiometry and we show that a determinant residue, which discriminates DciA- and DnaC/I-helicases, is critical in vivo. Our work is the first step toward the understanding of the ancestral mode of loading of bacterial replicative helicases on DNA. It sheds light on the strategy employed by phage helicase loaders to hijack bacterial replicative helicases and may explain the recurrent domestication of dnaC/I through evolution in bacteria.


Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , DnaB Helicases/química , Vibrio cholerae/enzimologia , Proteínas de Bactérias/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , DnaB Helicases/metabolismo , Modelos Moleculares , Conformação Proteica , Serina/química
17.
Nucleic Acids Res ; 49(12): 6863-6879, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34139017

RESUMO

Helicobacter pylori is a gram-negative, microaerophilic, pathogenic bacterium and a widespread colonizer of humans. H. pylori has developed mechanisms that enable it to overcome the harsh environment of the human stomach, including reactive oxygen species (ROS). Interestingly, up to now no typical regulator dedicated to the oxidative-stress response has been discovered. In this work, we reveal that the inhibitor of replication initiation HP1021 functions as a redox switch protein in H. pylori and plays an important role in response to oxidative stress of the gastric pathogen. Each of the two predicted HP1021 domains contains three cysteine residues. We show that the cysteine residues of HP1021 are sensitive to oxidation both in vitro and in vivo, and we demonstrate that HP1021 DNA-binding activity to oriC depends on the redox state of the protein. Moreover, Zn2+ modulates HP1021 affinity towards oriC template DNA. Transcription analysis of selected H. pylori genes by RT-qPCR indicated that HP1021 is directly involved in the oxygen-dependent control of H. pylori fecA3 and gluP genes, which are implicated in response to oxidative stress. In conclusion, HP1021 is a redox switch protein and could be a target for H. pylori control strategies.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Helicobacter pylori/genética , Estresse Oxidativo , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Cátions Bivalentes/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , Regulação Bacteriana da Expressão Gênica , Helicobacter pylori/metabolismo , Modelos Moleculares , Oxirredução , Ligação Proteica , Domínios Proteicos , Transcrição Genética
18.
Nat Commun ; 12(1): 3883, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162887

RESUMO

The Origin Recognition Complex (ORC) binds to sites in chromosomes to specify the location of origins of DNA replication. The S. cerevisiae ORC binds to specific DNA sequences throughout the cell cycle but becomes active only when it binds to the replication initiator Cdc6. It has been unclear at the molecular level how Cdc6 activates ORC, converting it to an active recruiter of the Mcm2-7 hexamer, the core of the replicative helicase. Here we report the cryo-EM structure at 3.3 Å resolution of the yeast ORC-Cdc6 bound to an 85-bp ARS1 origin DNA. The structure reveals that Cdc6 contributes to origin DNA recognition via its winged helix domain (WHD) and its initiator-specific motif. Cdc6 binding rearranges a short α-helix in the Orc1 AAA+ domain and the Orc2 WHD, leading to the activation of the Cdc6 ATPase and the formation of the three sites for the recruitment of Mcm2-7, none of which are present in ORC alone. The results illuminate the molecular mechanism of a critical biochemical step in the licensing of eukaryotic replication origins.


Assuntos
Proteínas de Ciclo Celular/genética , Replicação do DNA/genética , DNA Fúngico/genética , Complexo de Reconhecimento de Origem/genética , Origem de Replicação/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Microscopia Crioeletrônica , DNA Fúngico/química , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Complexo de Reconhecimento de Origem/química , Complexo de Reconhecimento de Origem/metabolismo , Ligação Proteica , Domínios Proteicos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
19.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34072703

RESUMO

Mercury (Hg) is a highly toxic and widespread pollutant. We previously reported that the exposure of Mytilus galloprovincialis for 24 h to doses of HgCl2 similar to those found in seawater (range 1-100 pM) produced alterations in the properties of protamine-like (PL) proteins that rendered them unable to bind and protect DNA from oxidative damage. In the present work, to deepen our studies, we analyzed PL proteins by turbidimetry and fluorescence spectroscopy and performed salt-induced release analyses of these proteins from sperm nuclei after the exposure of mussels to HgCl2 at the same doses. Turbidity assays indicated that mercury, at these doses, induced PL protein aggregates, whereas fluorescence spectroscopy measurements showed mercury-induced conformational changes. Indeed, the mobility of the PLII band changed in sodium dodecyl sulphate-polyacrylamide gel electrophoresis, particularly after exposure to 10-pM HgCl2, confirming the mercury-induced structural rearrangement. Finally, exposure to HgCl2 at all doses produced alterations in PL-DNA binding, detectable by DNA absorption spectra after the PL protein addition and by a decreased release of PLII and PLIII from the sperm nuclei. In conclusion, in this paper, we reported Hg-induced PL protein alterations that could adversely affect mussel reproductive activity, providing an insight into the molecular mechanism of Hg-related infertility.


Assuntos
Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mercúrio/farmacologia , Mytilus , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Animais , Núcleo Celular , Cromatina/química , Cromatina/genética , Proteínas de Ligação a DNA/química , Masculino , Cloreto de Mercúrio/farmacologia , Mercúrio/toxicidade , Água do Mar , Análise Espectral , Poluentes da Água/farmacologia , Poluentes da Água/toxicidade
20.
Nat Commun ; 12(1): 3335, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099661

RESUMO

Plants utilise intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors to detect pathogen effectors and activate local and systemic defence. NRG1 and ADR1 "helper" NLRs (RNLs) cooperate with enhanced disease susceptibility 1 (EDS1), senescence-associated gene 101 (SAG101) and phytoalexin-deficient 4 (PAD4) lipase-like proteins to mediate signalling from TIR domain NLR receptors (TNLs). The mechanism of RNL/EDS1 family protein cooperation is not understood. Here, we present genetic and molecular evidence for exclusive EDS1/SAG101/NRG1 and EDS1/PAD4/ADR1 co-functions in TNL immunity. Using immunoprecipitation and mass spectrometry, we show effector recognition-dependent interaction of NRG1 with EDS1 and SAG101, but not PAD4. An EDS1-SAG101 complex interacts with NRG1, and EDS1-PAD4 with ADR1, in an immune-activated state. NRG1 requires an intact nucleotide-binding P-loop motif, and EDS1 a functional EP domain and its partner SAG101, for induced association and immunity. Thus, two distinct modules (NRG1/EDS1/SAG101 and ADR1/EDS1/PAD4) mediate TNL receptor defence signalling.


Assuntos
Proteínas de Arabidopsis/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neuregulina-1/metabolismo , Imunidade Vegetal/fisiologia , Receptores Imunológicos/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Morte Celular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Imunidade Inata , Neuregulina-1/química , Neuregulina-1/genética , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Plantas Geneticamente Modificadas , Domínios Proteicos , Pseudomonas syringae , Receptores Imunológicos/química , Receptores Imunológicos/genética , Transdução de Sinais , Tabaco/genética , Tabaco/metabolismo
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