RESUMO
The UCS (UNC-45/CRO1/She4) chaperones play an evolutionarily conserved role in promoting myosin-dependent processes, including cytokinesis, endocytosis, RNA transport, and muscle development. To investigate the protein machinery orchestrating myosin folding and assembly, we performed a comprehensive analysis of Caenorhabditis elegans UNC-45. Our structural and biochemical data demonstrate that UNC-45 forms linear protein chains that offer multiple binding sites for cooperating chaperones and client proteins. Accordingly, Hsp70 and Hsp90, which bind to the TPR domain of UNC-45, could act in concert and with defined periodicity on captured myosin molecules. In vivo analyses reveal the elongated canyon of the UCS domain as a myosin-binding site and show that multimeric UNC-45 chains support organization of sarcomeric repeats. In fact, expression of transgenes blocking UNC-45 chain formation induces dominant-negative defects in the sarcomere structure and function of wild-type worms. Together, these findings uncover a filament assembly factor that directly couples myosin folding with myofilament formation.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Chaperonas Moleculares/metabolismo , Miofibrilas/metabolismo , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Modelos Moleculares , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Dobramento de Proteína , Estrutura Terciária de Proteína , Sarcômeros/metabolismoRESUMO
Eukaryotic cells are thought to arrange nucleosomes into extended arrays with evenly spaced nucleosomes phased at genomic landmarks. Here we tested to what extent this stereotypic organization describes the nucleosome organization in Saccharomyces cerevisiae using Fiber-Seq, a long-read sequencing technique that maps entire nucleosome arrays on individual chromatin fibers in a high throughput manner. With each fiber coming from a different cell, Fiber-Seq uncovers cell-to-cell heterogeneity. The long reads reveal the nucleosome architecture even over repetitive DNA such as the ribosomal DNA repeats. The absolute nucleosome occupancy, a parameter that is difficult to obtain with conventional sequencing approaches, is a direct readout of Fiber-Seq. We document substantial deviations from the stereotypical nucleosome organization with unexpectedly long linker DNAs between nucleosomes, gene bodies missing entire nucleosomes, cell-to-cell heterogeneity in nucleosome occupancy, heterogeneous phasing of arrays and irregular nucleosome spacing. Nucleosome array structures are indistinguishable throughout the gene body and with respect to the direction of transcription arguing against transcription promoting array formation. Acute nucleosome depletion destroyed most of the array organization indicating that nucleosome remodelers cannot efficiently pack nucleosomes under those conditions. Given that nucleosomes are cis-regulatory elements, the cell-to-cell heterogeneity uncovered by Fiber-Seq provides much needed information to understand chromatin structure and function.
Assuntos
Cromatina , Nucleossomos , Cromatina/genética , Nucleossomos/genética , DNA/genética , Genoma , Saccharomyces cerevisiae/genéticaRESUMO
DNA damage triggers chromatin remodeling by mechanisms that are poorly understood. The oncogene and chromatin remodeler ALC1/CHD1L massively decompacts chromatin in vivo yet is inactive prior to DNA-damage-mediated PARP1 induction. We show that the interaction of the ALC1 macrodomain with the ATPase module mediates auto-inhibition. PARP1 activation suppresses this inhibitory interaction. Crucially, release from auto-inhibition requires a poly-ADP-ribose (PAR) binding macrodomain. We identify tri-ADP-ribose as a potent PAR-mimic and synthetic allosteric effector that abrogates ATPase-macrodomain interactions, promotes an ungated conformation, and activates the remodeler's ATPase. ALC1 fragments lacking the regulatory macrodomain relax chromatin in vivo without requiring PARP1 activation. Further, the ATPase restricts the macrodomain's interaction with PARP1 under non-DNA damage conditions. Somatic cancer mutants disrupt ALC1's auto-inhibition and activate chromatin remodeling. Our data show that the NAD+-metabolite and nucleic acid PAR triggers ALC1 to drive chromatin relaxation. Modular allostery in this oncogene tightly controls its robust, DNA-damage-dependent activation.
Assuntos
Montagem e Desmontagem da Cromatina , Dano ao DNA , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias/enzimologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Regulação Alostérica , Sítios de Ligação , Linhagem Celular Tumoral , DNA Helicases/química , DNA Helicases/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Humanos , Mutação , Neoplasias/genética , Neoplasias/patologia , Conformação de Ácido Nucleico , Poli(ADP-Ribose) Polimerase-1/química , Poli(ADP-Ribose) Polimerase-1/genética , Poli ADP Ribosilação , Poli Adenosina Difosfato Ribose/química , Ligação Proteica , Relação Estrutura-Atividade , Fatores de TempoRESUMO
Specific recognition of cellular cargo and efficient transport to its correct intracellular destination is an infrastructural challenge faced by most eukaryotic cells. This remarkable deed is accomplished by processive motor proteins that are subject to robust regulatory mechanisms. The first level of regulation entails the ability of the motor to suppress its own activity. This autoinhibition is eventually relieved by specific cargo binding. To better understand the role of the cargo during motor activation, we dissected the activation mechanism of the ciliary homodimeric kinesin-2 from Caenorhabditis elegans by its physiological cargo. In functional reconstitution assays, we identified two cargo adaptor proteins that together are necessary and sufficient to allosterically activate the autoinhibited motor. Surprisingly, the orthologous adaptor proteins from the unicellular green algae Chlamydomonas reinhardtii also fully activated the kinesin-2 from worm, even though C. reinhardtii itself lacks a homodimeric kinesin-2 motor. The latter suggested that a motor activation mechanism similar to the C. elegans model existed already well before metazoans evolved, and prompted us to scrutinize predicted homodimeric kinesin-2 orthologs in other evolutionarily distant eukaryotes. We show that the ciliate Tetrahymena thermophila not only possesses a homodimeric kinesin-2 but that it also shares the same allosteric activation mechanism that we delineated in the C. elegans model. Our results point to a much more fundamental role of homodimeric kinesin-2 in intraflagellar transport (IFT) than previously thought and warrant further scrutiny of distantly related organisms toward a comprehensive picture of the IFT process and its evolution.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Cinesinas , Sequência de Aminoácidos , Animais , Transporte Biológico , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiologia , Cílios/metabolismo , Sequência Conservada , Flagelos/metabolismo , Cinesinas/genética , Cinesinas/fisiologiaRESUMO
Microbes have been coevolving with their host for millions of years, exploiting host resources to their own benefit. We show that viral and bacterial pathogens convergently evolved to hijack cellular mitogen-activated protein kinase (MAPK) p90-ribosomal S6-kinases (RSKs). Theiler's virus leader (L) protein binds RSKs and prevents their dephosphorylation, thus maintaining the kinases active. Recruitment of RSKs enables L-protein-mediated inhibition of eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2 or PKR) and stress granule formation. Strikingly, ORF45 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) and YopM protein of Yersinia use the same peptide motif as L to recruit and activate RSKs. All three proteins interact with a conserved surface-located loop of RSKs, likely acting as an allosteric regulation site. Some unrelated viruses and bacteria thus evolved to harness RSKs in a common fashion, yet to target distinct aspects of innate immunity. As documented for Varicella zoster virus ORF11, additional pathogens likely evolved to hijack RSKs, using a similar short linear motif.
Assuntos
Interações entre Hospedeiro e Microrganismos/fisiologia , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Bactérias/patogenicidade , Infecções Bacterianas/genética , Infecções Bacterianas/metabolismo , Evolução Biológica , Linhagem Celular , Regulação Viral da Expressão Gênica/genética , Interações entre Hospedeiro e Microrganismos/genética , Humanos , Proteínas Imediatamente Precoces/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Viroses/genética , Viroses/metabolismo , Replicação Viral/fisiologia , Vírus/patogenicidadeRESUMO
Nucleosome-nucleosome interactions drive the folding of nucleosomal arrays into dense chromatin fibers. A better physical account of the folding of chromatin fibers is necessary to understand the role of chromatin in regulating DNA transactions. Here, we studied the unfolding pathway of regular chromatin fibers as a function of single base pair increments in linker length, using both rigid base-pair Monte Carlo simulations and single-molecule force spectroscopy. Both computational and experimental results reveal a periodic variation of the folding energies due to the limited flexibility of the linker DNA. We show that twist is more restrictive for nucleosome stacking than bend, and find the most stable stacking interactions for linker lengths of multiples of 10 bp. We analyzed nucleosomes stacking in both 1- and 2-start topologies and show that stacking preferences are determined by the length of the linker DNA. Moreover, we present evidence that the sequence of the linker DNA also modulates nucleosome stacking and that the effect of the deletion of the H4 tail depends on the linker length. Importantly, these results imply that nucleosome positioning in vivo not only affects the phasing of nucleosomes relative to DNA but also directs the higher-order structure of chromatin.
Assuntos
Cromatina/química , DNA/química , Nucleossomos/química , Histonas/genética , Modelos Moleculares , Método de Monte Carlo , Conformação de Ácido NucleicoRESUMO
Pigment organelles, or melanosomes, are transported by kinesin, dynein, and myosin motors. As such, melanosome transport is an excellent model system to study the functional relationship between the microtubule- and actin-based transport systems. In mammalian melanocytes, it is well known that the Rab27a/melanophilin/myosin Va complex mediates actin-based transport in vivo. However, pathways that regulate the overall directionality of melanosomes on the actin/microtubule networks have not yet been delineated. Here, we investigated the role of PKA-dependent phosphorylation on the activity of the actin-based Rab27a/melanophilin/myosin Va transport complex in vitro. We found that melanophilin, specifically its C-terminal actin-binding domain (ABD), is a target of PKA. Notably, in vitro phosphorylation of the ABD closely recapitulated the previously described in vivo phosphorylation pattern. Unexpectedly, we found that phosphorylation of the ABD affected neither the interaction of the complex with actin nor its movement along actin tracks. Surprisingly, the phosphorylation state of melanophilin was instead important for reversible association with microtubules in vitro. Dephosphorylated melanophilin preferred binding to microtubules even in the presence of actin, whereas phosphorylated melanophilin associated with actin. Indeed, when actin and microtubules were present simultaneously, melanophilin's phosphorylation state enforced track selection of the Rab27a/melanophilin/myosin Va transport complex. Collectively, our results unmasked the regulatory dominance of the melanophilin adaptor protein over its associated motor and offer an unexpected mechanism by which filaments of the cytoskeletal network compete for the moving organelles to accomplish directional transport on the cytoskeleton in vivo.
Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Microtúbulos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Corantes Fluorescentes , Melanócitos/metabolismo , Melanossomas/metabolismo , Camundongos , Modelos Biológicos , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/metabolismo , Fosforilação , Domínios Proteicos , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas rab27 de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTP/metabolismoRESUMO
Two structurally distinct filamentous tracks, namely singlet microtubules in the cytoplasm and axonemes in the cilium, serve as railroads for long-range transport processes in vivo In all organisms studied so far, the kinesin-2 family is essential for long-range transport on axonemes. Intriguingly, in higher eukaryotes, kinesin-2 has been adapted to work on microtubules in the cytoplasm as well. Here, we show that heterodimeric kinesin-2 motors distinguish between axonemes and microtubules. Unlike canonical kinesin-1, kinesin-2 takes directional, off-axis steps on microtubules, but it resumes a straight path when walking on the axonemes. The inherent ability of kinesin-2 to side-track on the microtubule lattice restricts the motor to one side of the doublet microtubule in axonemes. The mechanistic features revealed here provide a molecular explanation for the previously observed partitioning of oppositely moving intraflagellar transport trains to the A- and B-tubules of the same doublet microtubule. Our results offer first mechanistic insights into why nature may have co-evolved the heterodimeric kinesin-2 with the ciliary machinery to work on the specialized axonemal surface for two-way traffic.
Assuntos
Proteínas de Anfíbios/química , Axonema/metabolismo , Cílios/metabolismo , Proteínas de Helminto/química , Cinesinas/química , Microtúbulos/metabolismo , Proteínas de Anfíbios/genética , Proteínas de Anfíbios/metabolismo , Animais , Axonema/ultraestrutura , Baculoviridae/genética , Baculoviridae/metabolismo , Transporte Biológico , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Cílios/ultraestrutura , Clonagem Molecular , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Cinesinas/genética , Cinesinas/metabolismo , Microtúbulos/ultraestrutura , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera/citologia , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
UNLABELLED: Protein crosslinking has been used for decades to derive structural information about proteins and protein complexes. Only recently, however, it became possible to map the amino acids involved in the crosslinks with the advent of high resolution mass spectrometry (MS). Here, we present Crossfinder, which automates the search for crosslinks formed by site-specifically incorporated crosslinking amino acids in LC-MS-MS data. AVAILABILITY AND IMPLEMENTATION: An executable version of Crossfinder for Windows machines (64-bit) is freely available to non-commercial users. It is bundled with a manual and example data.
Assuntos
Aminoácidos/química , Reagentes de Ligações Cruzadas/química , Complexos Multiproteicos/química , Proteínas/química , Espectrometria de Massas em Tandem/métodos , HumanosRESUMO
Nucleosome remodelling enzymes of the ISWI family reposition nucleosomes in eukaryotes. ISWI contains an ATPase and a HAND-SANT-SLIDE (HSS) domain. Conformational changes between these domains have been proposed to be critical for nucleosome repositioning by pulling flanking DNA into the nucleosome. We inserted flexible linkers at strategic sites in ISWI to disrupt this putative power stroke and assess its functional importance by quantitative biochemical assays. Notably, the flexible linkers did not disrupt catalysis. Instead of engaging in a power stroke, the HSS module might therefore assist DNA to ratchet into the nucleosome. Our results clarify the roles had by the domains and suggest that the HSS domain evolved to optimize a rudimentary remodelling engine.
Assuntos
Adenosina Trifosfatases/metabolismo , Nucleossomos/metabolismo , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Animais , Montagem e Desmontagem da Cromatina , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Nucleossomos/genética , Estrutura Terciária de Proteína , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
We present a strategy for rapidly gaining structural information about a protein from crosslinks formed by genetically encoded unnatural amino acids. We applied it to ISWI, a chromatin remodeling enzyme involved in chromatin assembly, DNA replication and transcription. ISWI is part of the vast Snf2 family of helicase-related proteins, many of which constitute the catalytic cores of chromatin remodeling complexes. Structural information about this family is scarce, hampering our mechanistic understanding of chromatin remodeling. Making use of cells that harbor a special tRNA/aminoacyl-tRNA synthetase pair, several residues within the ATPase domain of ISWI were individually substituted with the UV-reactive unnatural amino acid p-benzoyl-p-phenylalanine. Intramolecular crosslinks could be mapped with amino acid precision by high resolution tandem mass spectrometry and the novel bioinformatic tool "Crossfinder." Most crosslinks were fully consistent with published crystal structures of ISWI-related ATPases. A subset of crosslinks, however, disagreed with the conformations previously captured in crystal structures. We built a structural model using the distance information obtained from the crosslinks and the structure of the closest crystallized relative, Chd1. The model shows the ATPase lobes strongly rotated against each other, a movement postulated earlier to be necessary to achieve a catalytically competent state. The minimal requirements for solubility and protein amounts make our approach ideal for studying structures and conformations of proteins that are not amenable to conventional structural techniques.
Assuntos
Adenosina Trifosfatases/química , Proteínas de Drosophila/química , Fatores de Transcrição/química , Adenosina Trifosfatases/genética , Benzofenonas/química , Cromatografia Líquida , Proteínas de Drosophila/genética , Escherichia coli/genética , Modelos Moleculares , Conformação Molecular , Mutação , Fenilalanina/análogos & derivados , Fenilalanina/química , Estrutura Terciária de Proteína , Espectrometria de Massas em Tandem , Fatores de Transcrição/genética , Raios UltravioletaRESUMO
Nucleosomes are the basic compaction unit of chromatin and nucleosome structure and their higher-order assemblies regulate genome accessibility. Many post-translational modifications alter nucleosome dynamics, nucleosome-nucleosome interactions, and ultimately chromatin structure and gene expression. Here, we investigate the role of two post-translational modifications associated with actively transcribed regions, H3K36me3 and H4K5/8/12/16ac, in the contexts of tri-nucleosome arrays that provide a tractable model system for quantitative single-molecule analysis, while enabling us to probe nucleosome-nucleosome interactions. Direct visualization by AFM imaging reveals that H3K36me3 and H4K5/8/12/16ac nucleosomes adopt significantly more open and loose conformations than unmodified nucleosomes. Similarly, magnetic tweezers force spectroscopy shows a reduction in DNA outer turn wrapping and nucleosome-nucleosome interactions for the modified nucleosomes. The results suggest that for H3K36me3 the increased breathing and outer DNA turn unwrapping seen in mononucleosomes propagates to more open conformations in nucleosome arrays. In contrast, the even more open structures of H4K5/8/12/16ac nucleosome arrays do not appear to derive from the dynamics of the constituent mononucleosomes, but are driven by reduced nucleosome-nucleosome interactions, suggesting that stacking interactions can overrule DNA breathing of individual nucleosomes. We anticipate that our methodology will be broadly applicable to reveal the influence of other post-translational modifications and to observe the activity of nucleosome remodelers.
Assuntos
Histonas , Nucleossomos , Processamento de Proteína Pós-Traducional , Nucleossomos/metabolismo , Nucleossomos/química , Histonas/metabolismo , Histonas/química , Epigênese Genética , Código das Histonas , DNA/metabolismo , DNA/química , Conformação de Ácido Nucleico , Metilação , Microscopia de Força Atômica/métodosRESUMO
How chromatin enzymes work in condensed chromatin and how they maintain diffusional mobility inside remains unexplored. Here we investigated these challenges using the Drosophila ISWI remodeling ATPase, which slides nucleosomes along DNA. Folding of chromatin fibers did not affect sliding in vitro. Catalytic rates were also comparable in- and outside of chromatin condensates. ISWI cross-links and thereby stiffens condensates, except when ATP hydrolysis is possible. Active hydrolysis is also required for ISWI's mobility in condensates. Energy from ATP hydrolysis therefore fuels ISWI's diffusion through chromatin and prevents ISWI from cross-linking chromatin. Molecular dynamics simulations of a 'monkey-bar' model in which ISWI grabs onto neighboring nucleosomes, then withdraws from one before rebinding another in an ATP hydrolysis-dependent manner, qualitatively agree with our data. We speculate that monkey-bar mechanisms could be shared with other chromatin factors and that changes in chromatin dynamics caused by mutations in remodelers could contribute to pathologies.
RESUMO
Cilia are microtubule-based protrusions of the plasma membrane found on most eukaryotic cells. Their assembly is mediated through the conserved intraflagellar transport mechanism. One class of motor proteins involved in intraflagellar transport, kinesin-2, is unique among kinesin motors in that some of its members are composed of two distinct polypeptides. However, the biological reason for heterodimerization has remained elusive. Here we provide several interdependent reasons for the heterodimerization of the kinesin-2 motor KLP11/KLP20 of Caenorhabditis elegans cilia. One motor domain is unprocessive as a homodimer, but heterodimerization with a processive partner generates processivity. The "unprocessive" subunit is kept in this partnership as it mediates an asymmetric autoregulation of the motor activity. Finally, heterodimerization is necessary to bind KAP1, the in vivo link between motor and cargo.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Cinesinas/metabolismo , Movimento , Multimerização Proteica , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/química , Caenorhabditis elegans/citologia , Proteínas de Caenorhabditis elegans/química , Cílios/metabolismo , Cinesinas/química , Cinética , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
How chromatin enzymes work in condensed chromatin and how they maintain diffusional mobility inside remains unexplored. We investigated these challenges using the Drosophila ISWI remodeling ATPase, which slides nucleosomes along DNA. Folding of chromatin fibers did not affect sliding in vitro. Catalytic rates were also comparable in- and outside of chromatin condensates. ISWI cross-links and thereby stiffens condensates, except when ATP hydrolysis is possible. Active hydrolysis is also required for ISWI's mobility in condensates. Energy from ATP hydrolysis therefore fuels ISWI's diffusion through chromatin and prevents ISWI from cross-linking chromatin. Molecular dynamics simulations of a 'monkey-bar' model in which ISWI grabs onto neighboring nucleosomes, then withdraws from one before rebinding another in an ATP hydrolysis-dependent manner qualitatively agree with our data. We speculate that 'monkey-bar' mechanisms could be shared with other chromatin factors and that changes in chromatin dynamics caused by mutations in remodelers could contribute to pathologies.
RESUMO
Eukaryotes associate their genomes with histone proteins, forming nucleosome particles. Nucleosomes regulate and protect the genetic information. They often assemble into evenly spaced arrays of nucleosomes. These regular nucleosome arrays cover significant portions of the genome, in particular over genes. The presence of these evenly spaced nucleosome arrays is highly conserved throughout the entire eukaryotic domain. Here, we review the mechanisms behind the establishment of this primary structure of chromatin with special emphasis on the biogenesis of evenly spaced nucleosome arrays. We highlight the roles that transcription, nucleosome remodelers, DNA sequence, and histone density play towards the formation of evenly spaced nucleosome arrays and summarize our current understanding of their cellular functions. We end with key unanswered questions that remain to be explored to obtain an in-depth understanding of the biogenesis and function of the nucleosome landscape.
Assuntos
Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/química , DNA/química , Histonas/química , Nucleossomos/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA/genética , DNA/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Nucleossomos/química , Nucleossomos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Numerous chromatin remodeling enzymes position nucleosomes in eukaryotic cells. Aside from these factors, transcription, DNA sequence, and statistical positioning of nucleosomes also shape the nucleosome landscape. The precise contributions of these processes remain unclear due to their functional redundancy in vivo. By incisive genome engineering, we radically decreased their redundancy in Saccharomyces cerevisiae. The transcriptional machinery strongly disrupts evenly spaced nucleosomes. Proper nucleosome density and DNA sequence are critical for their biogenesis. The INO80 remodeling complex helps space nucleosomes in vivo and positions the first nucleosome over genes in an H2A.Z-independent fashion. INO80 requires its Arp8 subunit but unexpectedly not the Nhp10 module for spacing. Cells with irregularly spaced nucleosomes suffer from genotoxic stress including DNA damage, recombination and transpositions. We derive a model of the biogenesis of the nucleosome landscape and suggest that it evolved not only to regulate but also to protect the genome.
Assuntos
Cromatina , Epigenômica , Nucleossomos/fisiologia , Montagem e Desmontagem da Cromatina , DNA , Dano ao DNA , Engenharia , Células Eucarióticas , Proteínas de Grupo de Alta Mobilidade/metabolismo , Histonas , Proteínas dos Microfilamentos/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae , Fatores de TranscriçãoRESUMO
DNA topoisomerase II modulates DNA topology by relieving supercoil stress and by unknotting or decatenating entangled DNA. During its reaction cycle, the enzyme creates a transient double-strand break in one DNA segment, the G-DNA. This break serves as a gate through which another DNA segment is transported. Defined topoisomerase II cleavage sites in genomic and plasmid DNA have been previously mapped. To dissect the G-DNA recognition mechanism, we studied the affinity and reactivity of a series of DNA duplexes of varied sequence under conditions that only allow G-DNA to bind. These DNA duplexes could be cleaved to varying extents ranging from undetectable (<0.5%) to 80%. The sequence that defines a cleavage site resides within the central 20 bp of the duplex. The DNA affinity does not correlate with the ability of the enzyme to cleave DNA, suggesting that the binding step does not contribute significantly to the selection mechanism. Kinetic experiments show that the selectivity interactions are formed before rather than subsequent to cleavage. Presumably the binding energy of the cognate interactions is used to promote a conformational change that brings the enzyme into a cleavage competent state. The ability to modulate the extent of DNA cleavage by varying the DNA sequence may be valuable for future structural and mechanistic studies that aim to determine topoisomerase structures with DNA bound in pre- and post-cleavage states and to understand the conformational changes associated with DNA binding and cleavage.
Assuntos
DNA Topoisomerases Tipo II/metabolismo , DNA/metabolismo , Sítios de Ligação , DNA/química , DNA Topoisomerases Tipo II/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Cinética , Conformação Proteica , Especificidade por Substrato , TermodinâmicaRESUMO
ATP-dependent nucleosome remodeling factors sculpt the nucleosomal landscape of eukaryotic chromatin. They deposit or evict nucleosomes or reposition them along DNA in a process termed nucleosome sliding. Remodeling has traditionally been analyzed using mononucleosomes as a model substrate. In vivo, however, nucleosomes form extended arrays with regular spacing. Here, we describe how regularly spaced nucleosome arrays can be reconstituted in vitro and how these arrays can be used to dissect remodeling in the test tube. We outline two assays. The first assay senses various structural changes to a specific nucleosome within the nucleosomal array whereas the second assay is specific toward detecting repositioning of nucleosomes within the array. Both assays exploit changes to the accessibility of DNA to restriction enzymes during the remodeling reaction.
Assuntos
Montagem e Desmontagem da Cromatina , Eletroforese em Gel de Ágar/métodos , Nucleossomos/metabolismo , Animais , Drosophila , Histonas/metabolismo , Especificidade por SubstratoRESUMO
Chromatin remodeling factors assume critical roles by regulating access to nucleosomal DNA. To determine the architecture of the Drosophila ISWI remodeling enzyme, we developed an integrative structural approach that combines protein cross-linking, mass spectrometry, small-angle X-ray scattering, and computational modeling. The resulting structural model shows the ATPase module in a resting state with both ATPase lobes twisted against each other, providing support for a conformation that was recently trapped by crystallography. The autoinhibiting NegC region does not protrude from the ATPase module as suggested previously. The regulatory NTR domain is located near both ATPase lobes. The full-length enzyme is flexible and can adopt a compact structure in solution with the C-terminal HSS domain packing against the ATPase module. Our data imply a series of conformational changes upon activation of the enzyme and illustrate how the NTR, NegC, and HSS domains contribute to regulation of the ATPase module.