RESUMO
The Structural Maintenance of Chromosomes (SMC) protein complexes are DNA-binding molecular machines required to shape chromosomes into functional units and to safeguard the genome through cell division. These ring-shaped multi-subunit protein complexes, which are present in all kingdoms of life, achieve this by organizing chromosomes in three-dimensional space. Mechanistically, the SMC complexes hydrolyze ATP to either stably entrap DNA molecules within their lumen, or rapidly reel DNA into large loops, which allow them to link two stretches of DNA in cis or trans. In this chapter, the canonical structure of the SMC complexes is first introduced, followed by a description of the composition and general functions of the main types of eukaryotic and prokaryotic SMC complexes. Thereafter, the current model for how SMC complexes perform in vitro DNA loop extrusion is presented. Lastly, chromosome loop formation by SMC complexes is introduced, and how the DNA loop extrusion mechanism contributes to chromosome looping by SMC complexes in cells is discussed.
Assuntos
Cromossomos , Cromossomos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , DNA/química , DNA/metabolismo , DNA/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/química , Trifosfato de Adenosina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/químicaRESUMO
Biomolecules contain various heterogeneities in their structures and local chemical properties, and their functions emerge through the dynamics encoded by these heterogeneities. Molecular dynamics model-based studies will greatly contribute to the elucidation of such chemical/mechanical structure-dynamics-function relationships and the mechanisms that generate them. Coarse-grained molecular dynamics models with appropriately designed nonuniform local interactions play an important role in considering the various phenomena caused by large molecular complexes consisting of various proteins and DNA such as nuclear chromosomes. Therefore, in this chapter, we will introduce a method for constructing a coarse-grained molecular dynamics model that simulates the global behavior of each chromosome in the nucleus of a mammalian cell containing many giant chromosomes.
Assuntos
Núcleo Celular , Simulação de Dinâmica Molecular , Núcleo Celular/metabolismo , Núcleo Celular/química , Animais , Humanos , Cromossomos/química , DNA/química , DNA/metabolismo , MamíferosRESUMO
Beyond its role as the bearer of genetic material, DNA also plays a crucial role in the activation phase of innate immunity. Pathogen recognition receptors (PRRs) and their homologs, pathogen-associated molecular patterns (PAMPs), form the foundation for driving innate immune activation and the induction of immune responses during infection. In the context of DNA viruses or bacterial infections, specific DNA sequences are recognized and bound by DNA sensors, marking the DNA as a PAMP for host recognition and subsequent activation of innate immunity. The primary DNA sensor pathway known to date is cGAS-STING, which can induce Type I interferons (IFN) and innate immune responses against viruses and bacteria. Additionally, the cGAS-STING pathway has been identified to mediate functions in autophagy and senescence. Herein, we introduce methods for using DNA PAMPs as molecular tools to study the role of cGAS-STING and its signaling pathway in regulating innate immunity, both in vitro and in vivo.
Assuntos
DNA , Imunidade Inata , Proteínas de Membrana , Nucleotidiltransferases , Transdução de Sinais , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Humanos , DNA/metabolismo , DNA/genética , Animais , Moléculas com Motivos Associados a Patógenos/metabolismo , Moléculas com Motivos Associados a Patógenos/imunologia , CamundongosRESUMO
Studies of proteins, found in one of the most stress-resistant animals tardigrade Ramazzottius varieornatus, aim to reveal molecular principles of extreme tolerance to various types of stress and developing applications based on them for medicine, biotechnology, pharmacy, and space research. Tardigrade DNA/RNA-binding damage suppressor protein (Dsup) reduces DNA damage caused by reactive oxygen spices (ROS) produced upon irradiation and oxidative stresses in Dsup-expressing transgenic organisms. This work is focused on the determination of structural features of Dsup protein and Dsup-DNA complex, which refines details of protective mechanism. For the first time, intrinsically disordered nature of Dsup protein with highly flexible structure was experimentally proven and characterized by the combination of small angle X-ray scattering (SAXS) technique, circular dichroism spectroscopy, and computational methods. Low resolution models of Dsup protein and an ensemble of conformations were presented. In addition, we have shown that Dsup forms fuzzy complex with DNA.
Assuntos
DNA , Proteínas Intrinsicamente Desordenadas , Tardígrados , Animais , Tardígrados/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , DNA/metabolismo , DNA/química , Dano ao DNA , Espalhamento a Baixo Ângulo , Dicroísmo Circular , Modelos Moleculares , Ligação Proteica , Difração de Raios X , Conformação Proteica , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genéticaRESUMO
Accurate prediction of transcription factor binding sites (TFBSs) is essential for understanding gene regulation mechanisms and the etiology of diseases. Despite numerous advances in deep learning for predicting TFBSs, their performance can still be enhanced. In this study, we propose MLSNet, a novel deep learning architecture designed specifically to predict TFBSs. MLSNet innovatively integrates multisize convolutional fusion with long short-term memory (LSTM) networks to effectively capture DNA-sparse higher-order sequence features. Further, MLSNet incorporates super token attention and Bi-LSTM to systematically extract and integrate higher-order DNA shape features. Experimental results on 165 ChIP-seq (chromatin immunoprecipitation followed by sequencing) datasets indicate that MLSNet consistently outperforms several state-of-the-art algorithms in the prediction of TFBSs. Specifically, MLSNet reports average metrics: 0.8306 for ACC, 0.8992 for AUROC, and 0.9035 for AUPRC, surpassing the second-best methods by 1.82%, 1.68%, and 1.54%, respectively. This research delineates the effectiveness of combining multi-size convolutional layers with LSTM and DNA shape-based features in enhancing predictive accuracy. Moreover, this study comprehensively assesses the variability in model performance across different cell lines and transcription factors. The source code of MLSNet is available at https://github.com/minghaidea/MLSNet.
Assuntos
Aprendizado Profundo , Fatores de Transcrição , Fatores de Transcrição/metabolismo , Sítios de Ligação , Algoritmos , Biologia Computacional/métodos , Humanos , Sequenciamento de Cromatina por Imunoprecipitação/métodos , DNA/metabolismo , DNA/químicaRESUMO
Nucleotide excision repair (NER) is vital for genome integrity. Yet, our understanding of the complex NER protein machinery remains incomplete. Combining cryo-EM and XL-MS data with AlphaFold2 predictions, we build an integrative model of the NER pre-incision complex(PInC). Here TFIIH serves as a molecular ruler, defining the DNA bubble size and precisely positioning the XPG and XPF nucleases for incision. Using simulations and graph theoretical analyses, we unveil PInC's assembly, global motions, and partitioning into dynamic communities. Remarkably, XPG caps XPD's DNA-binding groove and bridges both junctions of the DNA bubble, suggesting a novel coordination mechanism of PInC's dual incision. XPA rigging interlaces XPF/ERCC1 with RPA, XPD, XPB, and 5' ssDNA, exposing XPA's crucial role in licensing the XPF/ERCC1 incision. Mapping disease mutations onto our models reveals clustering into distinct mechanistic classes, elucidating xeroderma pigmentosum and Cockayne syndrome disease etiology.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA , Endonucleases , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/química , Humanos , Endonucleases/metabolismo , Endonucleases/genética , Fator de Transcrição TFIIH/metabolismo , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/genética , Proteína Grupo D do Xeroderma Pigmentoso/metabolismo , Proteína Grupo D do Xeroderma Pigmentoso/genética , Proteína Grupo D do Xeroderma Pigmentoso/química , Microscopia Crioeletrônica , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Ligação Proteica , DNA/metabolismo , DNA/química , DNA/genética , Proteína de Replicação A/metabolismo , Proteína de Replicação A/genética , Modelos Moleculares , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Reparo por Excisão , Proteínas NuclearesRESUMO
Exploring the intricate interplay within and between nucleic acids, as well as their interactions with proteins, holds pivotal significance in unraveling the molecular complexities steering cancer initiation and progression. To investigate these interactions, a diverse array of highly specific and sensitive molecular techniques has been developed. The selection of a particular technique depends on the specific nature of the interactions. Typically, researchers employ an amalgamation of these different techniques to obtain a comprehensive and holistic understanding of inter- and intramolecular interactions involving DNA-DNA, RNA-RNA, DNA-RNA, or protein-DNA/RNA. Examining nucleic acid conformation reveals alternative secondary structures beyond conventional ones that have implications for cancer pathways. Mutational hotspots in cancer often lie within sequences prone to adopting these alternative structures, highlighting the importance of investigating intra-genomic and intra-transcriptomic interactions, especially in the context of mutations, to deepen our understanding of oncology. Beyond these intramolecular interactions, the interplay between DNA and RNA leads to formations like DNA:RNA hybrids (known as R-loops) or even DNA:DNA:RNA triplex structures, both influencing biological processes that ultimately impact cancer. Protein-nucleic acid interactions are intrinsic cellular phenomena crucial in both normal and pathological conditions. In particular, genetic mutations or single amino acid variations can alter a protein's structure, function, and binding affinity, thus influencing cancer progression. It is thus, imperative to understand the differences between wild-type (WT) and mutated (MT) genes, transcripts, and proteins. The review aims to summarize the frequently employed methods and techniques for investigating interactions involving nucleic acids and proteins, highlighting recent advancements and diverse adaptations of each technique.
Assuntos
DNA , Neoplasias , RNA , Humanos , RNA/genética , RNA/metabolismo , RNA/química , DNA/metabolismo , DNA/genética , DNA/química , Neoplasias/genética , Neoplasias/patologia , Neoplasias/metabolismo , Animais , Conformação de Ácido Nucleico , Proteínas/química , Proteínas/metabolismo , Proteínas/genética , MutaçãoRESUMO
BACKGROUND: Mutations occurring in nucleic acids or proteins may affect the binding affinities of protein-nucleic acid interactions. Although many efforts have been devoted to the impact of protein mutations, few computational studies have addressed the effect of nucleic acid mutations and explored whether the identical methodology could be applied to the prediction of binding affinity changes caused by these two mutation types. RESULTS: Here, we developed a generalized algorithm named PNBACE for both DNA and protein mutations. We first demonstrated that DNA mutations could induce varying degrees of changes in binding affinity from multiple perspectives. We then designed a group of energy-based topological features based on different energy networks, which were combined with our previous partition-based energy features to construct individual prediction models through feature selections. Furthermore, we created an ensemble model by integrating the outputs of individual models using a differential evolution algorithm. In addition to predicting the impact of single-point mutations, PNBACE could predict the influence of multiple-point mutations and identify mutations significantly reducing binding affinities. Extensive comparisons indicated that PNBACE largely performed better than existing methods on both regression and classification tasks. CONCLUSIONS: PNBACE is an effective method for estimating the binding affinity changes of protein-nucleic acid complexes induced by DNA or protein mutations, therefore improving our understanding of the interactions between proteins and DNA/RNA.
Assuntos
Algoritmos , DNA , Mutação , Ligação Proteica , DNA/metabolismo , Biologia Computacional/métodos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genéticaRESUMO
DNMT1 is an essential DNA methyltransferase that catalyzes the transfer of methyl groups to CpG islands in DNA and generates a prominent epigenetic mark. The catalytic activity of DNMT1 relies on its conformational plasticity and ability to change conformation from an auto-inhibited to an activated state. Here, we present four cryo-EM reconstructions of apo DNMT1 and DNTM1: non-productive DNA, DNTM1: H3Ub2-peptide, DNTM1: productive DNA complexes. Our structures demonstrate the flexibility of DNMT1's N-terminal regulatory domains during the transition from an apo 'auto-inhibited' to a DNA-bound 'non-productive' and finally a DNA-bound 'productive' state of DNMT1. Furthermore, we address the regulation of DNMT1's methyltransferase activity by a DNMT1-selective small-molecule inhibitor and ubiquitinated histone H3. We observe that DNMT1 binds DNA in a 'non-productive' state despite the presence of the inhibitor and present the cryo-EM reconstruction of full-length DNMT1 in complex with a di-ubiquitinated H3 peptide analogue. Taken together, our results provide structural insights into the reaction cycle of DNMT1.
Assuntos
Microscopia Crioeletrônica , DNA (Citosina-5-)-Metiltransferase 1 , DNA , Microscopia Crioeletrônica/métodos , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/química , DNA/metabolismo , DNA/química , Humanos , Histonas/metabolismo , Histonas/química , Modelos Moleculares , Ligação Proteica , Conformação Proteica , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/metabolismoRESUMO
Evolutionary annotation of genome maintenance (GM) proteins has conventionally been established by remote relationships within protein sequence databases. However, often no significant relationship can be established. Highly sensitive approaches to attain remote homologies based on iterative profile-to-profile methods have been developed. Still, these methods have not been systematically applied in the evolutionary annotation of GM proteins. Here, by applying profile-to-profile models, we systematically survey the repertoire of GM proteins from bacteria to man. We identify multiple GM protein candidates and annotate domains in numerous established GM proteins, among other PARP, OB-fold, Macro, TUDOR, SAP, BRCT, KU, MYB (SANT), and nuclease domains. We experimentally validate OB-fold and MIS18 (Yippee) domains in SPIDR and FAM72 protein families, respectively. Our results indicate that, surprisingly, despite the immense interest and long-term research efforts, the repertoire of genome stability caretakers is still not fully appreciated.
Assuntos
Domínios Proteicos , Humanos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Instabilidade Genômica , Evolução Molecular , DNA/química , DNA/metabolismo , Bases de Dados de Proteínas , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Modelos Moleculares , Anotação de Sequência Molecular , Bactérias/genética , Bactérias/metabolismoRESUMO
Collisions of the transcription and replication machineries on the same DNA strand can pose a significant threat to genomic stability. These collisions occur in part due to the formation of RNA-DNA hybrids termed R-loops, in which a newly transcribed RNA molecule hybridizes with the DNA template strand. This study investigated the role of RAD52, a known DNA repair factor, in preventing collisions by directing R-loop formation and resolution. We show that RAD52 deficiency increases R-loop accumulation, exacerbating collisions and resulting in elevated DNA damage. Furthermore, RAD52's ability to interact with the transcription machinery, coupled with its capacity to facilitate R-loop dissolution, highlights its role in preventing collisions. Lastly, we provide evidence of an increased mutational burden from double-strand breaks at conserved R-loop sites in human tumor samples, which is increased in tumors with low RAD52 expression. In summary, this study underscores the importance of RAD52 in orchestrating the balance between replication and transcription processes to prevent collisions and maintain genome stability.
Assuntos
Replicação do DNA , Instabilidade Genômica , Estruturas R-Loop , Proteína Rad52 de Recombinação e Reparo de DNA , Transcrição Gênica , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Replicação do DNA/genética , Estruturas R-Loop/genética , Humanos , Dano ao DNA , Quebras de DNA de Cadeia Dupla , DNA/metabolismo , DNA/genética , Reparo do DNA , Mutação , Neoplasias/genética , Neoplasias/metabolismoRESUMO
Extracellular vesicles (EVs) have emerged as key players in intercellular communication, facilitating the transfer of crucial cargo between cells. Liquid biopsy, particularly through the isolation of EVs, has unveiled a rich source of potential biomarkers for health and disease, encompassing proteins and nucleic acids. A milestone in this exploration occurred a decade ago with the identification of extracellular vesicle-associated DNA (EV-DNA) in the bloodstream of a patient diagnosed with pancreatic cancer. Subsequent years have witnessed substantial advancements, deepening our insights into the molecular intricacies of EV-DNA emission, detection, and analysis. Understanding the complexities surrounding the release of EV-DNA and addressing the challenges inherent in EV-DNA research are pivotal steps toward enhancing liquid biopsy-based strategies. These strategies, crucial for the detection and monitoring of various pathological conditions, particularly cancer, rely on a comprehensive understanding of why and how EV-DNA is released. In our review, we aim to provide a thorough summary of a decade's worth of research on EV-DNA. We will delve into diverse mechanisms of EV-DNA emission, its potential as a biomarker, its functional capabilities, discordant findings in the field, and the hurdles hindering its clinical application. Looking ahead to the next decade, we envision that advancements in EV isolation and detection techniques, coupled with improved standardization and data sharing, will catalyze the development of novel strategies exploiting EV-DNA as both a source of biomarkers and therapeutic targets.
Assuntos
DNA , Vesículas Extracelulares , Humanos , Vesículas Extracelulares/metabolismo , DNA/metabolismo , DNA/sangue , DNA/genética , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Biópsia Líquida/métodosRESUMO
Beyond its essential roles in ensuring faithful chromosome segregation and genomic stability, the human Smc5/6 complex acts as an antiviral factor. It binds to and impedes the transcription of extrachromosomal DNA templates; an ability which is lost upon integration of the DNA into the chromosome. How the complex distinguishes among different DNA templates is unknown. Here we show that, in human cells, Smc5/6 preferentially binds to circular rather than linear extrachromosomal DNA. We further demonstrate that the transcriptional process, per se, and particularly the accumulation of DNA secondary structures known to be substrates for topoisomerases, is responsible for Smc5/6 recruitment. More specifically, we find that in vivo Smc5/6 binds to positively supercoiled DNA. Those findings, in conjunction with our genome-wide Smc5/6 binding analysis showing that Smc5/6 localizes at few but highly transcribed chromosome loci, not only unveil a previously unforeseen role of Smc5/6 in DNA topology management during transcription but highlight the significance of sensing DNA topology as an antiviral defense mechanism.
Assuntos
Proteínas de Ciclo Celular , DNA Super-Helicoidal , Transcrição Gênica , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , DNA Super-Helicoidal/metabolismo , DNA Super-Helicoidal/genética , Ligação Proteica , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , DNA/metabolismo , DNA/genética , Conformação de Ácido Nucleico , DNA Circular/metabolismo , DNA Circular/genéticaRESUMO
DNA cleavage by nanomaterials has the potential to be utilized as an innovative tool for gene editing. Numerous nanomaterials exhibiting DNA cleavage properties have been identified and cataloged. Yet, the exploitation of property data through data-driven machine-learning approaches remains unexplored. A database was developed, compiling thirty distinctive characteristics, encompassing physical and chemical properties, as well as experimental conditions of nanomaterials that have demonstrated DNA cleavage capability such as in articles published over the past two decades. The DNA cleavage effect and efficiency of nanomaterials were predicted using machine learning algorithms such as support vector machines, deep neural networks, and random forest, and a classification accuracy of 0.93 for the cleavage effect was achieved. Moreover, the potential of utilizing larger datasets to enhance the predictive capacity of models was discussed. The findings indicate the feasibility of predicting nanomaterial properties based on experimental data. Evaluating the performance and effectiveness of the machine learning models trained using the existing data can furnish valuable insights for future materials research endeavors, especially for the design of DNA cleavage with specific sites.
Assuntos
Clivagem do DNA , Aprendizado de Máquina , Nanoestruturas , Nanoestruturas/química , Clivagem do DNA/efeitos dos fármacos , DNA/química , DNA/metabolismo , Redes Neurais de Computação , Máquina de Vetores de Suporte , AlgoritmosRESUMO
Synthesis and maturation of Okazaki Fragments is an incessant and highly efficient metabolic process completing the synthesis of the lagging strands at replication forks during S phase. Accurate Okazaki fragment maturation (OFM) is crucial to maintain genome integrity and, therefore, cell survival in all living organisms. In eukaryotes, OFM involves the consecutive action of DNA polymerase Pol ∂, 5' Flap endonuclease Fen1 and DNA ligase I, and constitutes the best example of a sequential process coordinated by the sliding clamp PCNA. For OFM to occur efficiently, cooperation of these enzymes with PCNA must be highly regulated. Here, we present evidence of a role for the K164-PCNA-deubiquitylase Ubp10 in the maturation of Okazaki fragments in the budding yeast Saccharomyces cerevisiae. We show that Ubp10 associates with lagging-strand DNA synthesis machineries on replicating chromatin to ensure timely ligation of Okazaki fragments by promoting PCNA dissociation from chromatin requiring lysine 164 deubiquitylation.
Assuntos
Cromatina , Replicação do DNA , Antígeno Nuclear de Célula em Proliferação , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Antígeno Nuclear de Célula em Proliferação/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Cromatina/metabolismo , DNA/metabolismo , Ubiquitinação , Endopeptidases/metabolismo , DNA Fúngico/metabolismo , DNA Fúngico/genética , Enzimas Desubiquitinantes/metabolismo , Endonucleases Flap/metabolismo , Endonucleases Flap/genética , DNA Ligase Dependente de ATP/metabolismo , DNA Ligase Dependente de ATP/genética , Ubiquitina TiolesteraseRESUMO
Fanzors are recently characterized RNA-guided DNA endonucleases found in eukaryotic organisms. In this issue of Cell, Xu, Saito et al. reveal the structural diversity of Fanzors and identify key features shared with TnpB and Cas12 proteins, providing a comprehensive perspective on their molecular function and evolution.
Assuntos
Sistemas CRISPR-Cas , Sistemas CRISPR-Cas/genética , Eucariotos/genética , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/genética , DNA/genética , DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , HumanosRESUMO
With recent advances in multi-color super-resolution light microscopy, it is possible to simultaneously visualize multiple subunits within biological structures at nanometer resolution. To optimally evaluate and interpret spatial proximity of stainings on such an image, colocalization analysis tools have to be able to integrate prior knowledge on the local geometry of the recorded biological complex. We present MultiMatch to analyze the abundance and location of chain-like particle arrangements in multi-color microscopy based on multi-marginal optimal unbalanced transport methodology. Our object-based colocalization model statistically addresses the effect of incomplete labeling efficiencies enabling inference on existent, but not fully observable particle chains. We showcase that MultiMatch is able to consistently recover existing chain structures in three-color STED images of DNA origami nanorulers and outperforms geometry-uninformed triplet colocalization methods in this task. MultiMatch generalizes to an arbitrary number of color channels and is provided as a user-friendly Python package comprising colocalization visualizations.
Assuntos
Processamento de Imagem Assistida por Computador , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Cor , Algoritmos , DNA/química , DNA/metabolismo , Microscopia/métodos , SoftwareRESUMO
Neural networks enable the processing of large, complex data sets with applications in disease diagnosis, cell profiling, and drug discovery. Beyond electronic computers, neural networks have been implemented using programmable biomolecules such as DNA; this confers unique advantages, such as greater portability, electricity-free operation, and direct analysis of patterns of biomolecules in solution. Analogous to bottlenecks in electronic computers, the computing power of DNA-based neural networks is limited by the ability to add more computing units, i.e., neurons. This limitation exists because current architectures require many nucleic acids to model a single neuron. Each additional neuron compounds existing problems such as long assembly times, high background signal, and cross-talk between components. Here, we test three strategies to solve this limitation and improve the scalability of DNA-based neural networks: (i) enzymatic synthesis for high-purity neurons, (ii) spatial patterning of neuron clusters based on their network position, and (iii) encoding neuron connectivity on a universal single-stranded DNA backbone. We show that neurons implemented via these strategies activate quickly, with a high signal-to-background ratio and process-weighted inputs. We rewired our modular neurons to demonstrate basic neural network motifs such as cascading, fan-in, and fan-out circuits. Finally, we designed a prototype two-layer microfluidic device to automate the operation of our circuits. We envision that our proposed design will help scale DNA-based neural networks due to its modularity, simplicity of synthesis, and compatibility with various neural network architectures. This will enable portable computing power for applications in portable diagnostics, compact data storage, and autonomous decision making for lab-on-a-chips.
Assuntos
DNA , Redes Neurais de Computação , DNA/química , DNA/metabolismo , Computadores Moleculares , Neurônios/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismoRESUMO
DNase II, identified in 1947 and named in 1953, is an acidic DNA endonuclease prevalent across organisms and crucial for normal growth. Despite its expression in nearly all human tissues, as well as its biological significance, DNase II's detailed functions and corresponding mechanisms remain unclear. Although many groups are trying to figure this out, progress is very limited. It is very hard to connect its indispensability with its DNA cleavage activity. In this study, we find that DNase II secreted to saliva can digest RNA in mildly acidic conditions, prompting us to hypothesize that salivary DNase II might digest RNA in the stomach. This finding is consistent with the interesting discovery reported in 1964 that RNA could inhibit DNase II's activity, which has been largely overlooked. This RNA digestion activity is further confirmed by using purified DNase II, showing activity to digest both DNA and RNA effectively. Here, we suggest redesignating DNase II as DNase II (RNase). The biological functions of DNase II are suggested to recycle intracellular RNA or digest external nucleic acids (both RNA and DNA) as nutrients. This discovery may untangle the mystery of DNase II and its significant biofunctions.
Assuntos
Endodesoxirribonucleases , RNA , RNA/metabolismo , Humanos , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , DNA/metabolismo , Saliva/metabolismo , Concentração de Íons de HidrogênioRESUMO
The human glycosylase OGG1 extrudes and excises the oxidized DNA base 8-oxoguanine (8-oxoG) to initiate base excision repair and plays important roles in many pathological conditions such as cancer, inflammation, and neurodegenerative diseases. Previous structural studies have used a truncated protein and short linear DNA, so it has been unclear how full-length OGG1 operates on longer DNA or on nucleosomes. Here we report cryo-EM structures of human OGG1 bound to a 35-bp long DNA containing an 8-oxoG within an unmethylated Cp-8-oxoG dinucleotide as well as to a nucleosome with an 8-oxoG at super-helical location (SHL)-5. The 8-oxoG in the linear DNA is flipped out by OGG1, consistent with previous crystallographic findings with a 15-bp DNA. OGG1 preferentially binds near dsDNA ends at the nucleosomal entry/exit sites. Such preference may underlie the enzyme's function in DNA double-strand break repair. Unexpectedly, we find that OGG1 bends the nucleosomal entry DNA, flips an undamaged guanine, and binds to internal nucleosomal DNA sites such as SHL-5 and SHL+6. We suggest that the DNA base search mechanism by OGG1 may be chromatin context-dependent and that OGG1 may partner with chromatin remodelers to excise 8-oxoG at the nucleosomal internal sites.