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1.
Mol Cell ; 79(6): 917-933.e9, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32755595

RESUMO

Despite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here we combine biochemical approaches and cryoelectron microscopy (cryo-EM) to visualize a cohesin loading intermediate in which DNA is locked between two gates that lead into the cohesin ring. Building on this structural framework, we design experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an N-terminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against the ATPase gate. ATP hydrolysis will lead to ATPase gate opening to complete DNA entry. Whether DNA loading is successful or results in loop extrusion might be dictated by a conserved kleisin N-terminal tail that guides the DNA through the kleisin gate. Our results establish the molecular basis for cohesin loading onto DNA.


Assuntos
Proteínas de Ciclo Celular/ultraestrutura , Cromátides/ultraestrutura , Proteínas Cromossômicas não Histona/ultraestrutura , DNA/ultraestrutura , Troca de Cromátide Irmã/genética , Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/genética , Cromátides/genética , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos/genética , Microscopia Crioeletrônica , DNA/genética , Conformação de Ácido Nucleico , Conformação Proteica , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestrutura
2.
Mol Cell ; 79(6): 902-916.e6, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32768407

RESUMO

A long-standing conundrum is how mitotic chromosomes can compact, as required for clean separation to daughter cells, while maintaining close parallel alignment of sister chromatids. Pursuit of this question, by high resolution 3D fluorescence imaging of living and fixed mammalian cells, has led to three discoveries. First, we show that the structural axes of separated sister chromatids are linked by evenly spaced "mini-axis" bridges. Second, when chromosomes first emerge as discrete units, at prophase, they are organized as co-oriented sister linear loop arrays emanating from a conjoined axis. We show that this same basic organization persists throughout mitosis, without helical coiling. Third, from prophase onward, chromosomes are deformed into sequential arrays of half-helical segments of alternating handedness (perversions), accompanied by correlated kinks. These arrays fluctuate dynamically over <15 s timescales. Together these discoveries redefine the foundation for thinking about the evolution of mitotic chromosomes as they prepare for anaphase segregation.


Assuntos
Proteínas de Ciclo Celular/genética , Cromossomos/genética , Proteínas de Ligação a DNA/genética , Mitose/genética , Adenosina Trifosfatases/genética , Anáfase/genética , Animais , Proteínas de Ciclo Celular/isolamento & purificação , Cromátides/genética , Proteínas Cromossômicas não Histona , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/isolamento & purificação , Imageamento Tridimensional , Mamíferos , Metáfase/genética , Prófase/genética
3.
Mol Cell ; 79(1): 3-5, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32619470

RESUMO

Kong et al. (2020) present the low-resolution structure of the ATPÉ£S-bound human condensin I and II complexes and demonstrate that human condensins can extrude DNA loops in a symmetric and asymmetric fashion and compact nucleosome-bound DNA.


Assuntos
Adenosina Trifosfatases/genética , Nucleossomos , Trifosfato de Adenosina , DNA , Proteínas de Ligação a DNA , Humanos , Complexos Multiproteicos
4.
PLoS Genet ; 16(7): e1008904, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32730253

RESUMO

The conserved ATPase, PCH-2/TRIP13, is required during both the spindle checkpoint and meiotic prophase. However, its specific role in regulating meiotic homolog pairing, synapsis and recombination has been enigmatic. Here, we report that this enzyme is required to proofread meiotic homolog interactions. We generated a mutant version of PCH-2 in C. elegans that binds ATP but cannot hydrolyze it: pch-2E253Q. In vitro, this mutant can bind a known substrate but is unable to remodel it. This mutation results in some non-homologous synapsis and impaired crossover assurance. Surprisingly, worms with a null mutation in PCH-2's adapter protein, CMT-1, the ortholog of p31comet, localize PCH-2 to meiotic chromosomes, exhibit non-homologous synapsis and lose crossover assurance. The similarity in phenotypes between cmt-1 and pch-2E253Q mutants suggest that PCH-2 can bind its meiotic substrates in the absence of CMT-1, in contrast to its role during the spindle checkpoint, but requires its adapter to hydrolyze ATP and remodel them.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Meiose/genética , Proteínas Nucleares/genética , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/genética , Animais , Caenorhabditis elegans/genética , Pareamento Cromossômico/genética , Segregação de Cromossomos/genética , Cromossomos/genética , Humanos , Mutação/genética , Fuso Acromático/genética
5.
Nucleic Acids Res ; 48(15): 8445-8460, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32644157

RESUMO

DNA lesions or other barriers frequently compromise replisome progress. The SF2 helicase RecG is a key enzyme in the processing of postreplication gaps or regressed forks in Escherichia coli. A deletion of the recG gene renders cells highly sensitive to a range of DNA damaging agents. Here, we demonstrate that RecG function is at least partially complemented by another SF2 helicase, RadD. A ΔrecGΔradD double mutant exhibits an almost complete growth defect, even in the absence of stress. Suppressors appear quickly, primarily mutations that compromise priA helicase function or recA promoter mutations that reduce recA expression. Deletions of uup (encoding the UvrA-like ABC system Uup), recO, or recF also suppress the ΔrecGΔradD growth phenotype. RadD and RecG appear to avoid toxic situations in DNA metabolism, either resolving or preventing the appearance of DNA repair intermediates produced by RecA or RecA-independent template switching at stalled forks or postreplication gaps. Barriers to replisome progress that require intervention by RadD or RecG occur in virtually every replication cycle. The results highlight the importance of the RadD protein for general chromosome maintenance and repair. They also implicate Uup as a new modulator of RecG function.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/genética , Reparo do DNA/genética , Replicação do DNA/genética , Proteínas de Escherichia coli/genética , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Mutação/genética , Recombinação Genética/genética
6.
Nucleic Acids Res ; 48(15): 8408-8430, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32663283

RESUMO

The chromatin remodelers SWI/SNF and RSC function in evicting promoter nucleosomes at highly expressed yeast genes, particularly those activated by transcription factor Gcn4. Ino80 remodeling complex (Ino80C) can establish nucleosome-depleted regions (NDRs) in reconstituted chromatin, and was implicated in removing histone variant H2A.Z from the -1 and +1 nucleosomes flanking NDRs; however, Ino80C's function in transcriptional activation in vivo is not well understood. Analyzing the cohort of Gcn4-induced genes in ino80Δ mutants has uncovered a role for Ino80C on par with SWI/SNF in evicting promoter nucleosomes and transcriptional activation. Compared to SWI/SNF, Ino80C generally functions over a wider region, spanning the -1 and +1 nucleosomes, NDR and proximal genic nucleosomes, at genes highly dependent on its function. Defects in nucleosome eviction in ino80Δ cells are frequently accompanied by reduced promoter occupancies of TBP, and diminished transcription; and Ino80 is enriched at genes requiring its remodeler activity. Importantly, nuclear depletion of Ino80 impairs promoter nucleosome eviction even in a mutant lacking H2A.Z. Thus, Ino80C acts widely in the yeast genome together with RSC and SWI/SNF in evicting promoter nucleosomes and enhancing transcription, all in a manner at least partly independent of H2A.Z editing.


Assuntos
Histonas/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcrição Genética , Ativação Transcricional/genética , Adenosina Trifosfatases/genética , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica/genética , Nucleossomos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/genética
7.
PLoS One ; 15(7): e0235033, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32639961

RESUMO

Lithium Chloride (LiCl) toxicity, mode of action and cellular responses have been the subject of active investigations over the past decades. In yeast, LiCl treatment is reported to reduce the activity and alters the expression of PGM2, a gene that encodes a phosphoglucomutase involved in sugar metabolism. Reduced activity of phosphoglucomutase in the presence of galactose causes an accumulation of intermediate metabolites of galactose metabolism leading to a number of phenotypes including growth defect. In the current study, we identify two understudied yeast genes, YTA6 and YPR096C that when deleted, cell sensitivity to LiCl is increased when galactose is used as a carbon source. The 5'-UTR of PGM2 mRNA is structured. Using this region, we show that YTA6 and YPR096C influence the translation of PGM2 mRNA.


Assuntos
Adenosina Trifosfatases/genética , Antimaníacos/farmacologia , Cloreto de Lítio/farmacologia , RNA Mensageiro/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Fosfoglucomutase/genética , Biossíntese de Proteínas/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética
8.
PLoS One ; 15(7): e0236520, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32730286

RESUMO

In eukaryotic cells, phospholipid flippases translocate phospholipids from the exoplasmic to the cytoplasmic leaflet of the lipid bilayer. Budding yeast contains five flippases, of which Cdc50p-Drs2p and Neo1p are primarily involved in membrane trafficking in endosomes and Golgi membranes. The ANY1/CFS1 gene was identified as a suppressor of growth defects in the neo1Δ and cdc50Δ mutants. Cfs1p is a membrane protein of the PQ-loop family and is localized to endosomal/Golgi membranes, but its relationship to phospholipid asymmetry remains unknown. The neo1Δ cfs1Δ mutant appears to function normally in membrane trafficking but may function abnormally in the regulation of phospholipid asymmetry. To identify a gene that is functionally relevant to NEO1 and CFS1, we isolated a mutation that is synthetically lethal with neo1Δ cfs1Δ and identified ERD1. Erd1p is a Golgi membrane protein that is involved in the transport of phosphate (Pi) from the Golgi lumen to the cytoplasm. The Neo1p-depleted cfs1Δ erd1Δ mutant accumulated plasma membrane proteins in the Golgi, perhaps due to a lack of phosphatidylinositol 4-phosphate. The Neo1p-depleted cfs1Δ erd1Δ mutant also exhibited abnormal structure of the endoplasmic reticulum (ER) and induced an unfolded protein response, likely due to defects in the retrieval pathway from the cis-Golgi region to the ER. Genetic analyses suggest that accumulation of Pi in the Golgi lumen is responsible for defects in Golgi functions in the Neo1p-depleted cfs1Δ erd1Δ mutant. Thus, the luminal ionic environment is functionally relevant to phospholipid asymmetry. Our results suggest that flippase-mediated phospholipid redistribution and luminal Pi concentration coordinately regulate Golgi membrane functions.


Assuntos
Complexo de Golgi/metabolismo , Fosfatos/metabolismo , Fosfolipídeos/metabolismo , Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/genética , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Mutação , Proteínas de Transferência de Fosfolipídeos/genética , Receptores Citoplasmáticos e Nucleares/genética , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Resposta a Proteínas não Dobradas
9.
Proc Natl Acad Sci U S A ; 117(31): 18608-18616, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690696

RESUMO

Transcription-coupled nucleotide excision repair (TC-NER) is an important DNA repair mechanism that removes RNA polymerase (RNAP)-stalling DNA damage from the transcribed strand (TS) of active genes. TC-NER deficiency in humans is associated with the severe neurological disorder Cockayne syndrome. Initiation of TC-NER is mediated by specific factors such as the human Cockayne syndrome group B (CSB) protein or its yeast homolog Rad26. However, the genome-wide role of CSB/Rad26 in TC-NER, particularly in the context of the chromatin organization, is unclear. Here, we used single-nucleotide resolution UV damage mapping data to show that Rad26 and its ATPase activity is critical for TC-NER downstream of the first (+1) nucleosome in gene coding regions. However, TC-NER on the transcription start site (TSS)-proximal half of the +1 nucleosome is largely independent of Rad26, likely due to high occupancy of the transcription initiation/repair factor TFIIH in this nucleosome. Downstream of the +1 nucleosome, the combination of low TFIIH occupancy and high occupancy of the transcription elongation factor Spt4/Spt5 suppresses TC-NER in Rad26-deficient cells. We show that deletion of SPT4 significantly restores TC-NER across the genome in a rad26∆ mutant, particularly in the downstream nucleosomes. These data demonstrate that the requirement for Rad26 in TC-NER is modulated by the distribution of TFIIH and Spt4/Spt5 in transcribed chromatin and Rad26 mainly functions downstream of the +1 nucleosome to remove TC-NER suppression by Spt4/Spt5.


Assuntos
Adenosina Trifosfatases , Reparo do DNA/genética , Nucleossomos/genética , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , DNA Helicases , Enzimas Reparadoras do DNA , Genoma Fúngico/genética , Humanos , Nucleossomos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
10.
Am J Hum Genet ; 107(2): 352-363, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32693025

RESUMO

MORC2 encodes an ATPase that plays a role in chromatin remodeling, DNA repair, and transcriptional regulation. Heterozygous variants in MORC2 have been reported in individuals with autosomal-dominant Charcot-Marie-Tooth disease type 2Z and spinal muscular atrophy, and the onset of symptoms ranges from infancy to the second decade of life. Here, we present a cohort of 20 individuals referred for exome sequencing who harbor pathogenic variants in the ATPase module of MORC2. Individuals presented with a similar phenotype consisting of developmental delay, intellectual disability, growth retardation, microcephaly, and variable craniofacial dysmorphism. Weakness, hyporeflexia, and electrophysiologic abnormalities suggestive of neuropathy were frequently observed but were not the predominant feature. Five of 18 individuals for whom brain imaging was available had lesions reminiscent of those observed in Leigh syndrome, and five of six individuals who had dilated eye exams had retinal pigmentary abnormalities. Functional assays revealed that these MORC2 variants result in hyperactivation of epigenetic silencing by the HUSH complex, supporting their pathogenicity. The described set of morphological, growth, developmental, and neurological findings and medical concerns expands the spectrum of genetic disorders resulting from pathogenic variants in MORC2.


Assuntos
Adenosina Trifosfatases/genética , Anormalidades Craniofaciais/genética , Transtornos do Crescimento/genética , Mutação/genética , Transtornos do Neurodesenvolvimento/genética , Fatores de Transcrição/genética , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Doenças Genéticas Inatas/genética , Heterozigoto , Humanos , Lactente , Deficiência Intelectual/genética , Masculino , Microcefalia/genética , Pessoa de Meia-Idade , Fenótipo , Adulto Jovem
11.
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
12.
No Shinkei Geka ; 48(6): 533-540, 2020 Jun.
Artigo em Japonês | MEDLINE | ID: mdl-32572006

RESUMO

Aplastic or twig-like middle cerebral artery(Ap/T-MCA)is a rare congenital anomaly, and several cases of ruptured cerebral aneurysm associated with Ap/T-MCA have been reported. Recently, the association of ring finger protein 213(RNF213)mutations with moyamoya disease has been identified, and the involvement of such mutations in intracranial arterial stenosis lesions other than those of moyamoya disease has been suggested. A 53-year-old woman with headache and nausea was admitted to our hospital. Computed tomography showed a diffuse subarachnoid hemorrhage. Cerebral angiography revealed left-sided Ap/T-MCA and two aneurysms in several fine arterioles. We performed trapping of these aneurysms. In the clinical course after surgery, she developed aphasia and mild motor paralysis. The patient was transferred to a rehabilitation hospital. The genetic screening revealed that she carried a heterozygous mutation of RNF213(c. 14429G>A p. R4810K). This is the first report of an association between Ap/T-MCA and RNF213 mutations. In patients with the RNF213 mutation, there is also the possibility of a progression of the intracranial arterial stenosis to other sites. Such patients should be carefully observed after the completion of their treatment.


Assuntos
Aneurisma Roto , Aneurisma Intracraniano , Doença de Moyamoya , Adenosina Trifosfatases/genética , Angiografia Cerebral , Feminino , Humanos , Pessoa de Meia-Idade , Artéria Cerebral Média , Mutação , Ubiquitina-Proteína Ligases/genética
13.
Nat Commun ; 11(1): 2887, 2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32513971

RESUMO

In eukaryotes, DNA wraps around histones to form nucleosomes, which are compacted into chromatin. DNA-templated processes, including transcription, require chromatin disassembly and reassembly mediated by histone chaperones. Additionally, distinct histone variants can replace core histones to regulate chromatin structure and function. Although replacement of H2A with the evolutionarily conserved H2A.Z via the SWR1 histone chaperone complex has been extensively studied, in plants little is known about how a reduction of H2A.Z levels can be achieved. Here, we show that NRP proteins cause a decrease of H2A.Z-containing nucleosomes in Arabidopsis under standard growing conditions. nrp1-1 nrp2-2 double mutants show an over-accumulation of H2A.Z genome-wide, especially at heterochromatic regions normally H2A.Z-depleted in wild-type plants. Our work suggests that NRP proteins regulate gene expression by counteracting SWR1, thereby preventing excessive accumulation of H2A.Z.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Montagem e Desmontagem da Cromatina/genética , Cromatina/genética , Histonas/genética , Chaperonas Moleculares/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cromatina/metabolismo , Regulação da Expressão Gênica de Plantas , Genoma de Planta/genética , Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Mutação , Nucleossomos/genética , Nucleossomos/metabolismo , Sequenciamento Completo do Genoma/métodos
14.
Proc Natl Acad Sci U S A ; 117(26): 14970-14977, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32541053

RESUMO

Msp1 is a conserved eukaryotic AAA+ ATPase localized to the outer mitochondrial membrane, where it is thought to extract mislocalized tail-anchored proteins. Despite recent in vivo and in vitro studies supporting this function, a mechanistic understanding of how Msp1 extracts its substrates is still lacking. Msp1's ATPase activity depends on its hexameric state, and previous characterizations of the cytosolic AAA+ domain in vitro had proved challenging due to its monomeric nature in the absence of the transmembrane domain. Here, we used a hexamerization scaffold to study the substrate-processing mechanism of the soluble Msp1 motor, the functional homo-hexameric state of which was confirmed by negative-stain electron microscopy. We demonstrate that Msp1 is a robust bidirectional protein translocase that is able to unfold diverse substrates by processive threading through its central pore. This unfoldase activity is inhibited by Pex3, a membrane protein proposed to regulate Msp1 at the peroxisome.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/genética , Humanos , Peroxissomos/genética , Peroxissomos/metabolismo , Domínios Proteicos , Dobramento de Proteína , Transporte Proteico
15.
Mem Inst Oswaldo Cruz ; 115: e190457, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32428081

RESUMO

BACKGROUND Imitation SWItch (ISWI) ATPase is the catalytic subunit in diverse chromatin remodeling complexes. These complexes modify histone-DNA interactions and therefore play a pivotal role in different DNA-dependent processes. In Trypanosoma cruzi, a protozoan that controls gene expression principally post-transcriptionally, the transcriptional regulation mechanisms mediated by chromatin remodeling are poorly understood. OBJECTIVE To characterise the ISWI remodeler in T. cruzi (TcISWI). METHODS A new version of pTcGW vectors was constructed to express green fluorescent protein (GFP)-tagged TcISWI. CRISPR-Cas9 system was used to obtain parasites with inactivated TcISWI gene and we determined TcISWI partners by cryomilling-affinity purification-mass spectrometry (MS) assay as an approximation to start to unravel the function of this protein. FINDINGS Our approach identified known ISWI partners [nucleoplasmin-like protein (NLP), regulator of chromosome condensation 1-like protein (RCCP) and phenylalanine/tyrosine-rich protein (FYRP)], previously characterised in T. brucei, and new components in TcISWI complex [DRBD2, DHH1 and proteins containing a domain characteristic of structural maintenance of chromosomes (SMC) proteins]. Data are available via ProteomeXchange with identifier PXD017869. MAIN CONCLUSIONS In addition to its participation in transcriptional silencing, as it was reported in T. brucei, the data generated here provide a framework that suggests a role for TcISWI chromatin remodeler in different nuclear processes in T. cruzi, including mRNA nuclear export control and chromatin compaction. Further work is necessary to clarify the TcISWI functional diversity that arises from this protein interaction study.


Assuntos
Adenosina Trifosfatases/genética , Montagem e Desmontagem da Cromatina/genética , Fatores de Transcrição/genética , Trypanosoma cruzi/genética , Animais , Western Blotting , Citometria de Fluxo , Regulação da Expressão Gênica
16.
Mol Cell ; 79(1): 127-139.e4, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32437639

RESUMO

C.neoformans Dnmt5 is an unusually specific maintenance-type CpG methyltransferase (DNMT) that mediates long-term epigenome evolution. It harbors a DNMT domain and SNF2 ATPase domain. We find that the SNF2 domain couples substrate specificity to an ATPase step essential for DNA methylation. Coupling occurs independent of nucleosomes. Hemimethylated DNA preferentially stimulates ATPase activity, and mutating Dnmt5's ATP-binding pocket disproportionately reduces ATPase stimulation by hemimethylated versus unmethylated substrates. Engineered DNA substrates that stabilize a reaction intermediate by mimicking a "flipped-out" conformation of the target cytosine bypass the SNF2 domain's requirement for hemimethylation. This result implies that ATP hydrolysis by the SNF2 domain is coupled to the DNMT domain conformational changes induced by preferred substrates. These findings establish a new role for a SNF2 ATPase: controlling an adjoined enzymatic domain's substrate recognition and catalysis. We speculate that this coupling contributes to the exquisite specificity of Dnmt5 via mechanisms related to kinetic proofreading.


Assuntos
Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , DNA Fúngico/metabolismo , Proteínas Fúngicas/metabolismo , Nucleossomos/metabolismo , Adenosina Trifosfatases/genética , Cryptococcus neoformans/genética , Cryptococcus neoformans/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA Fúngico/química , DNA Fúngico/genética , Proteínas Fúngicas/genética , Hidrólise , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
17.
Mol Cell ; 79(1): 99-114.e9, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32445620

RESUMO

Structural maintenance of chromosomes (SMC) complexes are essential for genome organization from bacteria to humans, but their mechanisms of action remain poorly understood. Here, we characterize human SMC complexes condensin I and II and unveil the architecture of the human condensin II complex, revealing two putative DNA-entrapment sites. Using single-molecule imaging, we demonstrate that both condensin I and II exhibit ATP-dependent motor activity and promote extensive and reversible compaction of double-stranded DNA. Nucleosomes are incorporated into DNA loops during compaction without being displaced from the DNA, indicating that condensin complexes can readily act upon nucleosome-bound DNA molecules. These observations shed light on critical processes involved in genome organization in human cells.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/química , DNA/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Nucleossomos/metabolismo , Adenosina Trifosfatases/genética , Proteínas de Ligação a DNA/genética , Humanos , Modelos Moleculares , Complexos Multiproteicos/genética , Ligação Proteica , Conformação Proteica , Imagem Individual de Molécula/métodos
18.
Curr Atheroscler Rep ; 22(4): 13, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32440785

RESUMO

PURPOSE OF REVIEW: Intracranial atherosclerosis (ICAS) is the most common cause of stroke throughout the world. It also increases the risk of recurrent stroke and dementia. As a complex and multifactorial disease, ICAS is influenced by multiple genetic, biological, and environmental factors. This review summarizes the candidate gene and genome-wide studies aimed at discovering genetic risk factors of ICAS. RECENT FINDINGS: Numerous studies have focused on the association between single-nucleotide polymorphisms (SNPs) of atherosclerosis-related genes and the risk of ICAS. Variants in adiponectin Q (ADIPOQ), ring finger protein 213 (RNF213), apolipoprotein E (APOE), phosphodiesterase 4D (PDE4D), methylenetetrahydrofolate reductase (MTHFR), lipoprotein lipase (LPL), α-adducin (ADD1) genes, angiotensin-converting enzyme (ACE), and other genes related to renin-angiotensin-aldosterone system have been associated with ICAS. We review the available evidences on the candidate genes and SNPs associated with genetic susceptibility to ICAS, and point out future developments of this field. Genetic discoveries could have clinical implications for intracranial atherosclerotic disease.


Assuntos
Predisposição Genética para Doença/genética , Arteriosclerose Intracraniana/genética , Adenosina Trifosfatases/genética , Adiponectina/genética , Apolipoproteínas E/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Feminino , Humanos , Lipase Lipoproteica/genética , Masculino , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Pessoa de Meia-Idade , Peptidil Dipeptidase A/genética , Polimorfismo de Nucleotídeo Único , Fatores de Risco , Ubiquitina-Proteína Ligases/genética
19.
J Biosci Bioeng ; 130(2): 149-158, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32414665

RESUMO

The hyperthermophilic archaeon Thermococcus kodakarensis can grow on pyruvate or maltooligosaccharides through H2 fermentation. H2 production levels of members of the Thermococcales are high, and studies to improve their production potential have been reported. Although H2 production is primary metabolism, here we aimed to partially uncouple cell growth and H2 production of T. kodakarensis. Additional A1-type ATPase genes were introduced into T. kodakarensis KU216 under the control of two promoters; the strong constitutive cell surface glycoprotein promoter, Pcsg, and the sugar-inducible fructose-1,6-bisphosphate aldolase promoter, Pfba. Whereas cells with the A1-type ATPase genes under the control of Pcsg displayed only trace levels of growth, cells with Pfba (strain KUA-PF) displayed growth sufficient for further analysis. Increased levels of A1-type ATPase protein were detected in KUA-PF cells grown on pyruvate or maltodextrin, when compared to the levels in the host strain KU216. The growth and H2 production levels of strain KUA-PF with pyruvate or maltodextrin as a carbon and electron source were analyzed and compared to those of the host strain KU216. Compared to a small decrease in total H2 production, significantly larger decreases in cell growth were observed, resulting in an increase in cell-specific H2 production. Quantification of the substrate also revealed that ATPase overexpression led to increased cell-specific pyruvate and maltodextrin consumptions. The results clearly indicate that ATPase production results in partial uncoupling of cell growth and H2 production in T. kodakarensis.


Assuntos
Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Regulação da Expressão Gênica em Archaea , Hidrogênio/metabolismo , Thermococcus/enzimologia , Thermococcus/genética , Carbono/metabolismo , Dosagem de Genes/fisiologia , Regulação da Expressão Gênica em Archaea/genética , Organismos Geneticamente Modificados/metabolismo , Polissacarídeos/metabolismo , Ácido Pirúvico/metabolismo
20.
Pol J Pathol ; 71(1): 69-74, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32429658

RESUMO

Molecular next gene sequencing was used to evaluate mutations in 409 common mutated cancer-related genes in malignant mesothelioma of tunica vaginalis testis (MMTVT) of 81-year-old man. Multifocal papillary-solid areas contained necrosis among highly cellular fields with multiple mitoses. It was positive for WT1, CKAE1/AE3, calretinin, CK7 with negativity for CK5, PSA, TTF-1. Following mutations were revealed in PARP1 (NM_001618: c.2285TG, p.K135R), MTRR (NM_024010: c.147A>G, p.I49M) and two sorts of mutations in structure of KMT2C gene (NM_170606: c.2447_2448insA (c.2447dupA), p.Y816fs and NM_170606: c.1042G>A, p.D348N) for the first time in MMTVT.


Assuntos
Mesotelioma/genética , Neoplasias Testiculares/genética , Adenosina Trifosfatases/genética , Idoso de 80 Anos ou mais , Proteínas de Ligação a DNA/genética , Ferredoxina-NADP Redutase/genética , Humanos , Masculino , Mutação , Fator de Transcrição PAX8/genética , Poli(ADP-Ribose) Polimerase-1/genética , Ubiquitina-Proteína Ligases/genética
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