RESUMO
Structural characterization of chromatin is challenging due to conformational and compositional heterogeneity in vivo and dynamic properties that limit achievable resolution in vitro. Although the maximum resolution for solving structures of large macromolecular assemblies by electron microscopy has recently undergone profound increases, X-ray crystallographic approaches may still offer advantages for certain systems. One such system is compact chromatin, wherein the crystalline state recapitulates the crowded molecular environment within the nucleus. Here we show that nucleosomal constructs with cohesive-ended DNA can be designed that assemble into different types of circular configurations or continuous fibers extending throughout crystals. We demonstrate the utility of the method for characterizing nucleosome compaction and linker histone binding at near-atomic resolution but also advance its application for tackling further problems in chromatin structural biology and for generating novel types of DNA nanostructures. We provide a library of cohesive-ended DNA fragment expression constructs and a strategy for engineering DNA-based nanomaterials with a seemingly vast potential variety of architectures and histone chemistries.
Assuntos
DNA/química , Nucleossomos/química , Animais , Pareamento de Bases , Bioengenharia , Galinhas , Cromatina/química , Cristalografia por Raios X , Histonas/química , Humanos , Modelos MolecularesRESUMO
Targeting defined histone protein sites in chromatin is an emerging therapeutic approach that can potentially be enhanced by allosteric effects within the nucleosome. Here we characterized a novel hetero-bimetallic compound with a design based on a nucleosomal allostery effect observed earlier for two unrelated drugs-the RuII antimetastasis/antitumor RAPTA-T and the AuI anti-arthritic auranofin. The RuII moiety binds specifically to two H2A glutamate residues on the nucleosome acidic patch, allosterically triggering a cascade of structural changes that promote binding of the AuI moiety to selective histidine residues on H3, resulting in cross-linking sites that are over 35â Å distant. By tethering the H2A-H2B dimers to the H3-H4 tetramer, the hetero-bimetallic compound significantly increases stability of the nucleosome, illustrating its utility as a site-selective cross-linking agent.
Assuntos
Reagentes de Ligações Cruzadas/química , Nucleossomos/química , Nucleossomos/metabolismo , Sítio Alostérico , Modelos Moleculares , Estrutura MolecularRESUMO
Platinum-based anticancer drugs act therapeutically by forming DNA adducts, but suffer from severe toxicity and resistance problems, which have not been overcome in spite of decades of research. And yet defined chromatin targets have generally not been considered in the drug development process. Here we designed novel platinum-intercalator species to target a highly deformed DNA site near the nucleosome center. Between two seemingly similar structural isomers, we find a striking difference in DNA site selectivity in vitro, which comes about from stereochemical constraints that limit the reactivity of the trans isomer to special DNA sequence elements while still allowing the cis isomer to efficiently form adducts at internal sites in the nucleosome core. This gives the potential for controlling nucleosome site targeting in vivo, which would engender sensitivity to epigenetic distinctions and in particular cell type/status-dependent differences in nucleosome positioning. Moreover, while both compounds yield very similar DNA-adduct structures and display antitumor cell activity rivalling that of cisplatin, the cis isomer, relative to the trans, has a much more rapid cytotoxic effect and distinct impact on cell function. The novel stereochemical principles for controlling DNA site selectivity we discovered could aid in the design of improved site discriminating agents.
Assuntos
Antineoplásicos/química , Substâncias Intercalantes/química , Naftalimidas/química , Nucleossomos/química , Compostos Organoplatínicos/química , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Adutos de DNA/análise , Humanos , Substâncias Intercalantes/toxicidade , Naftalimidas/toxicidade , Compostos Organoplatínicos/toxicidade , EstereoisomerismoRESUMO
Understanding how small molecules interact with DNA is essential since it underlies a multitude of pathological conditions and therapeutic interventions. Many different intercalator compounds have been studied because of their activity as mutagens or drugs, but little is known regarding their interaction with nucleosomes, the protein-packaged form of DNA in cells. Here, using crystallographic methods and molecular dynamics simulations, we discovered that adducts formed by [(η(6) -THA)Ru(ethylenediamine)Cl][PF6 ] (THA=5,8,9,10-tetrahydroanthracene; RAED-THA-Cl[PF6 ]) in the nucleosome comprise a novel one-stranded intercalation and DNA distortion mode. Conversely, the THA group in fact remains solvent exposed and does not disrupt base stacking in RAED-THA adducts on B-form DNA. This newly observed DNA binding mode and topology dependence may actually be prevalent and should be considered when studying covalently binding intercalating compounds.
Assuntos
Antracenos/química , DNA/química , Simulação de Dinâmica Molecular , Compostos Organometálicos/química , Sítios de Ligação , DNA/metabolismo , Etilenodiaminas/química , Substâncias Intercalantes/químicaRESUMO
Because of the widespread epigenetic changes ensuing from carcinogenesis, structural and chemical features of chromatin provide unique targets for developing safer and more effective anticancer drugs. Metal-based agents have a potential advantage over other small molecular species in that characteristics of coordination geometry, redox state and ligand exchange allow one to fine-tune reactivity and affinity properties in a distinct fashion. This intersection of chromatin biology and bioinorganic medicinal chemistry is the subject of multiple collaborations in and between Switzerland and Singapore.
Assuntos
Antineoplásicos/química , Química Farmacêutica/história , Cisplatino/química , Adutos de DNA/química , Metais/química , Neoplasias/química , Animais , Antineoplásicos/história , Antineoplásicos/farmacologia , Química Farmacêutica/instrumentação , Química Farmacêutica/métodos , Cromatina/efeitos dos fármacos , Cromatina/ultraestrutura , Cisplatino/farmacologia , Complexos de Coordenação/química , Complexos de Coordenação/história , Complexos de Coordenação/farmacologia , Cristalografia por Raios X , Adutos de DNA/ultraestrutura , História do Século XXI , Humanos , Cooperação Internacional , Neoplasias/tratamento farmacológico , Oxirredução , Singapura , SuíçaRESUMO
Chromatin organization and composition impart sophisticated regulatory features critical to eukaryotic genomic function. Although DNA sequence-dependent histone octamer binding is important for nucleosome activity, many aspects of this phenomenon have remained elusive. We studied nucleosome structure and stability with diverse DNA sequences, including Widom 601 derivatives with the highest known octamer affinities, to establish a simple model behind the mechanics of sequence dependency. This uncovers the unique but unexpected role of TA dinucleotides and a propensity for G|C-rich sequence elements to conform energetically favourably at most locations around the histone octamer, which rationalizes G|C% as the most predictive factor for nucleosome occupancy in vivo. In addition, our findings reveal dominant constraints on double helix conformation by H3-H4 relative to H2A-H2B binding and DNA sequence context-dependency underlying nucleosome structure, positioning and stability. This provides a basis for improved prediction of nucleosomal properties and the design of tailored DNA constructs for chromatin investigations.
Assuntos
DNA/química , Histonas/química , Nucleossomos/química , Animais , Sequência de Bases , Fenômenos Biomecânicos , Modelos Moleculares , Conformação de Ácido Nucleico , Xenopus laevisRESUMO
Heavy metal compounds have toxic and medicinal potential through capacity to form strong specific bonds with macromolecules, and the interaction of platinum drugs at the major groove nitrogen atom of guanine bases primarily underlies their therapeutic activity. By crystallographic analysis of transition metal-and in particular platinum compound-DNA site selectivity in the nucleosome core, we establish that steric accessibility, which is controlled by specific structural parameters of the double helix, modulates initial guanine-metal bond formation. Moreover, DNA conformational features can be linked to both similarities and distinctions in platinum drug adduct formation between the naked and nucleosomal DNA states. Notably, structures that facilitate initial platinum-guanine bond formation can oppose cross-link generation, rationalizing the occurrence of long-lived therapeutically ineffective monofunctional adducts. These findings illuminate DNA structure-dependent reactivity and provide a novel framework for understanding metal-double helix interactions, which should facilitate the development of improved chromatin-targeting medicinal agents.
Assuntos
Antineoplásicos/química , Reagentes de Ligações Cruzadas/química , Adutos de DNA/química , Compostos Organoplatínicos/química , Compostos de Platina/química , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Nucleossomos/químicaRESUMO
Understanding the molecular mechanisms behind regulation of chromatin folding through covalent modifications of the histone N-terminal tails is hampered by a lack of accessible chromatin containing precisely modified histones. We study the internal folding and intermolecular self-association of a chromatin system consisting of saturated 12-mer nucleosome arrays containing various combinations of completely acetylated lysines at positions 5, 8, 12 and 16 of histone H4, induced by the cations Na(+), K(+), Mg(2+), Ca(2+), cobalt-hexammine(3+), spermidine(3+) and spermine(4+). Histones were prepared using a novel semi-synthetic approach with native chemical ligation. Acetylation of H4-K16, but not its glutamine mutation, drastically reduces cation-induced folding of the array. Neither acetylations nor mutations of all the sites K5, K8 and K12 can induce a similar degree of array unfolding. The ubiquitous K(+), (as well as Rb(+) and Cs(+)) showed an unfolding effect on unmodified arrays almost similar to that of H4-K16 acetylation. We propose that K(+) (and Rb(+)/Cs(+)) binding to a site on the H2B histone (R96-L99) disrupts H4K16 ε-amino group binding to this specific site, thereby deranging H4 tail-mediated nucleosome-nucleosome stacking and that a similar mechanism operates in the case of H4-K16 acetylation. Inter-array self-association follows electrostatic behavior and is largely insensitive to the position or nature of the H4 tail charge modification.
Assuntos
Histonas/química , Nucleossomos/química , Acetilação , Cátions/química , Cromatina/química , Simulação por Computador , Histonas/genética , Histonas/metabolismo , Lisina/metabolismo , Mutação , Conformação de Ácido Nucleico , Potássio/química , Conformação Proteica , Eletricidade EstáticaRESUMO
Heavy metals have the potential to engage in strong bonding interactions and can thus function in essential as well as toxic or therapeutic capacities. We conducted crystallographic analyses of heavy cation binding to the nucleosome core particle and found that Co(2+) and Ni(2+) preferentially associate with the DNA major groove, in a sequence- and conformation-dependent manner. Conversely, Rb(+) and Cs(+) are found to bind only opportunistically to minor groove elements of the DNA, in particular at narrow AT dinucleotide sites. Furthermore, relative to Mn(2+) the aggressive coordination of Co(2+) and Ni(2+) to guanine bases is observed to induce a shift in histone-DNA register around the nucleosome center by stabilizing DNA stretching over one region accompanied by expulsion of two bases at an opposing location. These 'softer' transition metals also associate with multiple histone protein sites, including inter-nucleosomal cross-linking, and display a proclivity for coordination to histidine. Sustained binding and the ability to induce structural perturbations at specific locations in the nucleosome may contribute to genetic and epigenetic mechanisms of carcinogenesis mediated by Co(2+) and Ni(2+).
Assuntos
Metais Pesados/química , Nucleossomos/química , Animais , Sequência de Bases , Cátions Monovalentes/química , Cobalto/química , DNA/química , Histidina/química , Histonas/química , Modelos Moleculares , Dados de Sequência Molecular , Níquel/química , Purinas/química , Xenopus laevisRESUMO
DNA stretching in the nucleosome core can cause dramatic structural distortions, which may influence compaction and factor recognition in chromatin. We find that the base pair unstacking arising from stretching-induced extreme minor groove kinking near the nucleosome centre creates a hot spot for intercalation and alkylation by a novel anticancer compound. This may have far reaching implications for how chromatin structure can influence binding of intercalator species and indicates potential for the development of site selective DNA-binding agents that target unique conformational features of the nucleosome.
Assuntos
Antineoplásicos Alquilantes/química , DNA/química , Compostos de Epóxi/química , Substâncias Intercalantes/química , Naftalimidas/química , Nucleossomos/química , Cristalografia por Raios X , Pegada de DNA , Modelos Moleculares , Conformação de Ácido NucleicoRESUMO
X-ray crystallographic and biochemical investigation of the reaction of cisplatin and oxaliplatin with nucleosome core particle and naked DNA reveals that histone octamer association can modulate DNA platination. Adduct formation also occurs at specific histone methionine residues, which could serve as a nuclear platinum reservoir influencing adduct transfer to DNA. Our findings suggest that the nucleosome center may provide a favorable target for the design of improved platinum anticancer drugs.
Assuntos
Antineoplásicos/química , Platina/química , Sequência de Aminoácidos , Sequência de Bases , Cristalografia por Raios X , Adutos de DNA/química , Histonas/química , Histonas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Nucleossomos/química , Nucleossomos/metabolismo , Estrutura Terciária de Proteína , Sensibilidade e EspecificidadeRESUMO
Chromosome structure at the multi-nucleosomal level has remained ambiguous in spite of its central role in epigenetic regulation and genome dynamics. Recent investigations of chromatin architecture portray diverse modes of interaction within and between nucleosome chains, but how this is realized at the atomic level is unclear. Here we present near-atomic resolution crystal structures of nucleosome fibres that assemble from cohesive-ended dinucleosomes with and without linker histone. As opposed to adopting folded helical '30 nm' structures, the fibres instead assume open zigzag conformations that are interdigitated with one another. Zigzag conformations obviate extreme bending of the linker DNA, while linker DNA size (nucleosome repeat length) dictates fibre configuration and thus fibre-fibre packing, which is supported by variable linker histone binding. This suggests that nucleosome chains have a predisposition to interdigitate with specific characteristics under condensing conditions, which rationalizes observations of local chromosome architecture and the general heterogeneity of chromatin structure.
Assuntos
Nucleossomos/química , Nucleossomos/metabolismo , Sequência de Bases , Cromatina/química , Cromatina/metabolismo , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Modelos Moleculares , Conformação Molecular , Ligação ProteicaRESUMO
Nucleosome positioning and reorganization regulate DNA site exposure in chromatin. Platinum anticancer agents form DNA adducts that disrupt nuclear activities, triggering apoptosis. Mechanistic insight would aid in the development of improved therapies to circumvent drug toxicity and resistance. We show that platinum adducts formed by reaction of cisplatin or oxaliplatin with the nucleosome core inhibit histone octamer-DNA sliding but do not cause significant alteration of positioning. Thus, adduct formation reinforces positional preferences intrinsic to the DNA sequence, which indicates that modulation of platinum drug site selectivity by histone octamer association may relate to nucleosome-specific properties of DNA. This sheds light on platinum drug-mediated inhibition of chromatin remodeling in vivo and suggests that adducts can shield their own repair and interfere with genomic activities by directly altering nucleosome dynamics.
Assuntos
Nucleossomos/química , Platina/química , Modelos Moleculares , Conformação MolecularRESUMO
The poly(ADP-ribose) polymerase, PARP1, plays a key role in maintaining genomic integrity by detecting DNA damage and mediating repair. γH2A.X is the primary histone marker for DNA double-strand breaks and PARP1 localizes to H2A.X-enriched chromatin damage sites, but the basis for this association is not clear. We characterize the kinetics of PARP1 binding to a variety of nucleosomes harbouring DNA double-strand breaks, which reveal that PARP1 associates faster with (γ)H2A.X- versus H2A-nucleosomes, resulting in a higher affinity for the former, which is maximal for γH2A.X-nucleosome that is also the activator eliciting the greatest poly-ADP-ribosylation catalytic efficiency. The enhanced activities with γH2A.X-nucleosome coincide with increased accessibility of the DNA termini resulting from the H2A.X-Ser139 phosphorylation. Indeed, H2A- and (γ)H2A.X-nucleosomes have distinct stability characteristics, which are rationalized by mutational analysis and (γ)H2A.X-nucleosome core crystal structures. This suggests that the γH2A.X epigenetic marker directly facilitates DNA repair by stabilizing PARP1 association and promoting catalysis.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Reparo do DNA/genética , Histonas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Nucleossomos/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/isolamento & purificação , Biocatálise , Cristalografia por Raios X , Quebras de DNA de Cadeia Dupla , Epigênese Genética , Histonas/síntese química , Histonas/ultraestrutura , Modelos Moleculares , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/isolamento & purificação , Nucleossomos/ultraestrutura , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/isolamento & purificação , Poli ADP Ribosilação/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
DNA stretching in chromatin may facilitate its compaction and influence site recognition by nuclear factors. In vivo, stretching has been estimated to occur at the equivalent of one to two base-pairs (bp) per nucleosome. We have determined the crystal structure of a nucleosome core particle containing 145 bp of DNA (NCP145). Compared to the structure with 147 bp, the NCP145 displays two incidences of stretching one to two double-helical turns from the particle dyad axis. The stretching illustrates clearly a mechanism for shifting DNA position by displacement of a single base-pair while maintaining nearly identical histone-DNA interactions. Increased DNA twist localized to a short section between adjacent histone-DNA binding sites advances the rotational setting, while a translational component involves DNA kinking at a flanking region that initiates elongation by unstacking bases. Furthermore, one stretched region of the NCP145 displays an extraordinary 55 degrees kink into the minor groove situated 1.5 double-helical turns from the particle dyad axis, a hot spot for gene insertion by HIV-integrase, which prefers highly distorted substrate. This suggests that nucleosome position and context within chromatin could promote extreme DNA kinking that may influence genomic processes.
Assuntos
DNA/química , Modelos Moleculares , Conformação de Ácido Nucleico , Nucleossomos/química , Animais , Sequência de Bases , Histonas/genética , Humanos , Dados de Sequência Molecular , Xenopus laevisRESUMO
Architectural proteins that reconfigure the paths of DNA segments are required for the establishment of functional interfaces in many genomic transactions. A single-chain derivative of the DNA architectural protein integration host factor was found to adopt two stable conformational states in complex with a specific DNA target. In the so-called open state, the degree of protein-induced DNA bending is reduced significantly compared with the closed state. The conformational switch between these states is controlled by divalent metal binding in two electronegative zones arising from the lysine-to-glutamate substitution in the protein body proximal to the phosphate backbone of one DNA arm. We show that this switch can be employed to control the efficiency of site-specific recombination catalyzed by lambda integrase. Introduction of acidic residues at the protein-DNA interface holds potential for the design of metal-mediated switches for the investigation of functional relationships.
Assuntos
Proteínas de Ligação a DNA/química , DNA/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Cátions Bivalentes , Cristalografia por Raios X , DNA/genética , DNA Super-Helicoidal/química , DNA Super-Helicoidal/genética , Proteínas de Ligação a DNA/genética , Fatores Hospedeiros de Integração/química , Fatores Hospedeiros de Integração/genética , Substâncias Macromoleculares , Proteínas de Membrana/química , Proteínas de Membrana/genética , Microscopia de Força Atômica , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido NucleicoRESUMO
Exploitation of drug-drug synergism and allostery could yield superior therapies by capitalizing on the immensely diverse, but highly specific, potential associated with the biological macromolecular landscape. Here we describe a drug-drug synergy mediated by allosteric cross-talk in chromatin, whereby the binding of one drug alters the activity of the second. We found two unrelated drugs, RAPTA-T and auranofin, that yield a synergistic activity in killing cancer cells, which coincides with a substantially greater number of chromatin adducts formed by one of the compounds when adducts from the other agent are also present. We show that this occurs through an allosteric mechanism within the nucleosome, whereby defined histone adducts of one drug promote reaction of the other drug at a distant, specific histone site. This opens up possibilities for epigenetic targeting and suggests that allosteric modulation in nucleosomes may have biological relevance and potential for therapeutic interventions.
Assuntos
Cromatina/metabolismo , Sinergismo Farmacológico , Regulação Alostérica/efeitos dos fármacos , Auranofina/química , Auranofina/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cristalografia por Raios X , Histonas/química , Histonas/metabolismo , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Nucleossomos/metabolismo , Compostos Organometálicos/química , Compostos Organometálicos/farmacologia , Estrutura Secundária de ProteínaRESUMO
Cisplatin is a widely used anti-cancer drug, but its effect is often limited by acquired resistance to the compound during treatment. Here, we use a combination of transmission electron microscopy (TEM) and nanoscale-secondary ion mass spectrometry (NanoSIMS) to reveal differences between cisplatin uptake in human ovarian cancers cells, which are known to be susceptible to acquired resistance to cisplatin. Both cisplatin sensitive and resistant cell lines were studied, revealing markedly less cisplatin in the resistant cell line. In cisplatin sensitive cells, Pt was seen to distribute diffusely in the cells with hotspots in the nucleolus, mitochondria, and autophagosomes. Inductively coupled plasma mass spectrometry (ICP-MS) was used to validate the NanoSIMS results.
Assuntos
Antineoplásicos/metabolismo , Cisplatino/metabolismo , Resistencia a Medicamentos Antineoplásicos , Microscopia Eletrônica de Transmissão/métodos , Neoplasias Ovarianas/metabolismo , Espectrometria de Massa de Íon Secundário/métodos , Antineoplásicos/farmacologia , Cisplatino/farmacologia , Feminino , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Frações Subcelulares/patologia , Células Tumorais CultivadasRESUMO
The 'acidic patch' is a highly electronegative cleft on the histone H2A-H2B dimer in the nucleosome. It is a fundamental motif for protein binding and chromatin dynamics, but the cellular impact of targeting this potentially therapeutic site with exogenous molecules remains unclear. Here, we characterize a family of binuclear ruthenium compounds that selectively target the nucleosome acidic patch, generating intra-nucleosomal H2A-H2B cross-links as well as inter-nucleosomal cross-links. In contrast to cisplatin or the progenitor RAPTA-C anticancer drugs, the binuclear agents neither arrest specific cell cycle phases nor elicit DNA damage response, but rather induce an irreversible, anomalous state of condensed chromatin that ultimately results in apoptosis. In vitro, the compounds induce misfolding of chromatin fibre and block the binding of the regulator of chromatin condensation 1 (RCC1) acidic patch-binding protein. This family of chromatin-modifying molecules has potential for applications in drug development and as tools for chromatin research.