Nucleoprotein Complexes Research Articles

Atomic-Resolution Structure of the Protein Encoded by Gene V of fd Bacteriophage in Complex with Viral ssDNA Determined by Magic-Angle Spinning Solid-State NMR.

F-specific filamentous phages, elongated particles with circular single-stranded DNA encased in a symmetric protein capsid, undergo an intermediate step, where thousands of homodimers of a non-structural protein, gVp, bind to newly synthesized strands of DNA, preventing further DNA replication and preparing the circular genome in an elongated conformation for assembly of a new virion structure at the membrane. While the structure of the free homodimer is known, the ssDNA-bound conformation has yet to be determined. We report an atomic-resolution structure of the gVp monomer bound to ssDNA of fd phage in the nucleoprotein complex elucidated via magic-angle spinning solid-state NMR. The model presents significant conformational changes with respect to the free form. These modifications facilitate the binding mechanism and possibly promote cooperative binding in the assembly of the gVp-ssDNA complex.

SARS-CoV-2 Exploits Non-Canonical Autophagic Processes to Replicate, Mature, and Egress the Infected Vero E6 Cells.

The coronavirus transforms the cytoplasm of susceptible cells to support virus replication. It also activates autophagy-like processes, the role of which is not well understood. Here, we studied SARS-CoV-2-infected Vero E6 cells using transmission electron microscopy and autophagy PCR array. After 6-24 h post-infection (hpi), the cytoplasm of infected cells only contained double-membrane vesicles, phagophores, and phagosomes engulfing virus particles and cytoplasmic debris, including damaged mitochondria. The phagosomes interacted with the viral nucleoprotein complex, virus particles, mitochondria, and lipid droplets. The phagosomes transformed into egress vacuoles, which broke through the plasmalemma and discharged the virus particles. The Vero E6 cells exhibited pronounced virus replication at 6 hpi, which stabilized at 18-24 hpi at a high level. The autophagy PCR array tests revealed a significant upregulation of 10 and downregulation of 8 autophagic gene markers out of 84. Altogether, these results underline the importance of autophagy-like processes for SARS-CoV-2 maturation and egress, and point to deviations from a canonical autophagy response.

Structural transitions during the cooperative assembly of baculovirus single-stranded DNA-binding protein on ssDNA.

Single-stranded DNA-binding proteins (SSBs) interact with single-stranded DNA (ssDNA) to form filamentous structures with various degrees of cooperativity, as a result of intermolecular interactions between neighboring SSB subunits on ssDNA. However, it is still challenging to perform structural studies on SSB-ssDNA filaments at high resolution using the most studied SSB models, largely due to the intrinsic flexibility of these nucleoprotein complexes. In this study, HaLEF-3, an SSB protein from Helicoverpa armigera nucleopolyhedrovirus, was used for in vitro assembly of SSB-ssDNA filaments, which were structurally studied at atomic resolution using cryo-electron microscopy. Combined with the crystal structure of ssDNA-free HaLEF-3 octamers, our results revealed that the three-dimensional rearrangement of HaLEF-3 induced by an internal hinge-bending movement is essential for the formation of helical SSB-ssDNA complexes, while the contacting interface between adjacent HaLEF-3 subunits remains basically intact. We proposed a local cooperative SSB-ssDNA binding model, in which, triggered by exposure to oligonucleotides, HaLEF-3 molecules undergo ring-to-helix transition to initiate continuous SSB-SSB interactions along ssDNA. Unique structural features revealed by the assembly of HaLEF-3 on ssDNA suggest that HaLEF-3 may represent a new class of SSB.

Rapid Single-Pot Assembly of Modular Chromatin Proteins for Epigenetic Engineering.

Chromatin is the nucleoprotein complex that organizes genomic DNA in the nuclei of eukaryotic cells. Chromatin-modifying enzymes and chromatin-binding regulators generate chromatin states that affect DNA compaction, repair, gene expression, and ultimately cell phenotype. Many natural chromatin mediators contain subdomains that can be isolated and recombined to build synthetic regulators and probes. Engineered chromatin proteins make up a growing collection of new tools for cell engineering and can help deepen our understanding of the mechanism by which chromatin features, such as modifications of histones and DNA, contribute to the epigenetic states that govern DNA-templated processes. To support efficient exploration of the large combinatorial design space of synthetic chromatin proteins, we have developed a Golden Gate assembly method for one-step construction of protein-encoding recombinant DNA. A set of standard 2-amino acid linkers allows facile assembly of any combination of up to four protein modules, obviating the need to design different compatible overhangs to ligate different modules. Beginning with the identification of protein modules of interest, a synthetic chromatin protein can be built and expressed in vitro or in cells in under 2weeks.

High-throughput imaging and quantitative analysis uncovers the nature of plasmid positioning by ParABS.

The faithful segregation and inheritance of bacterial chromosomes and low-copy number plasmids requires dedicated partitioning systems. The most common of these, ParABS, consists of ParA, a DNA-binding ATPase and ParB, a protein that binds to centromeric-like <i>parS</i> sequences on the DNA cargo. The resulting nucleoprotein complexes are believed to move up a self-generated gradient of nucleoid-associated ParA. However, it remains unclear how this leads to the observed cargo positioning and dynamics. In particular, the evaluation of models of plasmid positioning has been hindered by the lack of quantitative measurements of plasmid dynamics. Here, we use high-throughput imaging, analysis and modelling to determine the dynamical nature of these systems. We find that F plasmid is actively brought to specific subcellular home positions within the cell with dynamics akin to an over-damped spring. We develop a unified stochastic model that quantitatively explains this behaviour and predicts that cells with the lowest plasmid concentration transition to oscillatory dynamics. We confirm this prediction for F plasmid as well as a distantly-related ParABS system. Our results indicate that ParABS regularly positions plasmids across the nucleoid but operates just below the threshold of an oscillatory instability, which according to our model, minimises ATP consumption. Our work also clarifies how various plasmid dynamics are achievable in a single unified stochastic model. Overall, this work uncovers the dynamical nature of plasmid positioning by ParABS and provides insights relevant for chromosome-based systems.

The Domino Effect: Nucleosome Dynamics and the Regulation of Base Excision Repair Enzymes

DNA damage is induced by exogenous and endogenous sources, creating a variety of lesions. However, the cellular repair machinery that addresses and corrects this damage must contend with the fact that genomic DNA is sequestered in the nucleoprotein complex of chromatin. As the minimal unit of DNA compaction, the nucleosome core particle (NCP) is a major determinant of repair and poses unique barriers to DNA accessibility. This review outlines how the base excision repair (BER) pathway is modulated by the NCP and describes the structural and dynamic factors that influence the ability of BER enzymes to find and repair damage. Structural characteristics of the NCP such as nucleobase positioning and occupancy will be explored along with factors that impact the dynamic nature of NCPs to increase mobilization of nucleosomal DNA. We will discuss how altering the dynamics of NCPs initiates a domino effect that results in the regulation of BER enzymes.

Formation of synthetic RNA protein granules using engineered phage-coat-protein -RNA complexes

Liquid-solid transition, also known as gelation, is a specific form of phase separation in which molecules cross-link to form a highly interconnected compartment with solid – like dynamical properties. Here, we utilize RNA hairpin coat-protein binding sites to form synthetic RNA based gel-like granules via liquid-solid phase transition. We show both in-vitro and in-vivo that hairpin containing synthetic long non-coding RNA (slncRNA) molecules granulate into bright localized puncta. We further demonstrate that upon introduction of the coat-proteins, less-condensed gel-like granules form with the RNA creating an outer shell with the proteins mostly present inside the granule. Moreover, by tracking puncta fluorescence signals over time, we detected addition or shedding events of slncRNA-CP nucleoprotein complexes. Consequently, our granules constitute a genetically encoded storage compartment for protein and RNA with a programmable controlled release profile that is determined by the number of hairpins encoded into the RNA. Our findings have important implications for the potential regulatory role of naturally occurring granules and for the broader biotechnology field.

Infrared nanospectroscopic imaging of DNA molecules on mica surface

Significant efforts have been done in last two decades to develop nanoscale spectroscopy techniques owning to their great potential for single-molecule structural detection and in addition, to resolve open questions in heterogeneous biological systems, such as protein–DNA complexes. Applying IR-AFM technique has become a powerful leverage for obtaining simultaneous absorption spectra with a nanoscale spatial resolution for studied proteins, however the AFM-IR investigation of DNA molecules on surface, as a benchmark for a nucleoprotein complexes nanocharacterization, has remained elusive. Herein, we demonstrate methodological approach for acquisition of AFM-IR mapping modalities with corresponding absorption spectra based on two different DNA deposition protocols on spermidine and Ni2+ pretreated mica surface. The nanoscale IR absorbance of distinctly formed DNA morphologies on mica are demonstrated through series of AFM-IR absorption maps with corresponding IR spectrum. Our results thus demonstrate the sensitivity of AFM-IR nanospectroscopy for a nucleic acid research with an open potential to be employed in further investigation of nucleoprotein complexes.

Lipid Surfaces and Glutamate Anions Enhance Formation of Dynamic Biomolecular Condensates Containing Bacterial Cell Division Protein FtsZ and Its DNA-Bound Regulator SlmA.

Dynamic biomolecular condensates formed by liquid-liquid phase separation can regulate the spatial and temporal organization of proteins, thus modulating their functional activity in cells. Previous studies showed that the cell division protein FtsZ from Escherichia coli formed dynamic phase-separated condensates with nucleoprotein complexes containing the FtsZ spatial regulator SlmA under crowding conditions, with potential implications for condensate-mediated spatiotemporal control of FtsZ activity in cell division. In the present study, we assessed formation of these condensates in the presence of lipid surfaces and glutamate ions to better approximate the E. coli intracellular environment. We found that potassium glutamate substantially promoted the formation of FtsZ-containing condensates when compared to potassium chloride in crowded solutions. These condensates accumulated on supported lipid bilayers and eventually fused, resulting in a time-dependent increase in the droplet size. Moreover, the accumulated condensates were dynamic, capturing protein from the external phase. FtsZ partitioned into the condensates at the lipid surface only in its guanosine diphosphate (GDP) form, regardless of whether it came from FtsZ polymer disassembly upon guanosine triphosphate (GTP) exhaustion. These results provide insights into the behavior of these GTP-responsive condensates in minimal membrane systems, which suggest how these membraneless assemblies may tune critical bacterial division events during the cell cycle.

Anti-CRISPR proteins function through thermodynamic tuning and allosteric regulation of CRISPR RNA-guided surveillance complex.

CRISPR RNA-guided detection and degradation of foreign DNA is a dynamic process. Viruses can interfere with this cellular defense by expressing small proteins called anti-CRISPRs. While structural models of anti-CRISPRs bound to their target complex provide static snapshots that inform mechanism, the dynamics and thermodynamics of these interactions are often overlooked. Here, we use hydrogen deuterium exchange-mass spectrometry (HDX-MS) and differential scanning fluorimetry (DSF) experiments to determine how anti-CRISPR binding impacts the conformational landscape of the type IF CRISPR RNA guided surveillance complex (Csy) upon binding of two different anti-CRISPR proteins (AcrIF9 and AcrIF2). The results demonstrate that AcrIF2 binding relies on enthalpic stabilization, whereas AcrIF9 uses an entropy driven reaction to bind the CRISPR RNA-guided surveillance complex. Collectively, this work reveals the thermodynamic basis and mechanistic versatility of anti-CRISPR-mediated immune suppression. More broadly, this work presents a striking example of how allosteric effectors are employed to regulate nucleoprotein complexes.

P497 Candida DNA extraction from positive blood culture bottles

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMBackgroundMolecular technique of detecting candida from blood is a quick method to identify candidemia. A major limitation of the molecular method is the difficulty associated with breaking fungal cell wall, the DNA extraction step still requires more than half of a working day.The successful extraction of DNA involves effective disruption of cells, denaturation of protein, and nucleoprotein complexes, and inactivation of nucleases such as DNase. The extracted DNA should have low contamination of proteins.ObjectivesTo perform DNA extraction using phenol-chloroform method.2. To speciate candida using PCR-RFLP.Methods Candida DNA is isolated using phenol-chloroform method. The PCR products of different candida species obtained directly from blood samples were subjected to restriction enzyme (MspI).ResultsDNA was extracted from 50 samples, out of which 40 samples were from known positive blood culture bottles and the remaining 10 were known negative for candida. Of the 40 known positive blood culture bottles, 34 produced bands of various molecular weights and all 10 negative cultures did not show bands. Representative amplicon is shown in the gel picture (Figure 1).Out of 40 candida-positive blood culture samples, 7 species have been identified using PCR- RFLP method.ConclusionThe turn-around time can be highly reduced by extracting DNA from positive blood culture bottles and then performing PCR and RFLP for speciation compared with the conventional method.

Nontraditional Method for Telomere Staining by PNA Probes.

The standardFISH uses DNA probes to hybridize to the designated complementary strands. This is DNA-DNA interaction, and it usually takes much longer time to obtain detectable signals compared to other reactions such as immunochemical reactions and simple chemical reactions. Certain proteins bind to specific DNA sequences and regulate the biological function of DNA. These DNA-binding proteins have specific domains to interact with single- or double-stranded DNA. Some of telomere proteins apparently bind to telomere sequence and form nucleoprotein complex to protect chromosome ends. Using telomere PNA probes, probes can be accumulated at the telomere sites in a non-hybridization manner. This chapter introduces nontraditional PNA telomere staining protocol without DNA-DNA hybridization to visualize telomere locations on metaphase chromosomes.

Telomere Length as a Marker of Biological Aging: A Critical Review of Recent Literature.

Aging is characterized as a syndrome of deleterious, progressive, universal, and irreversible function changes affecting every structural and functional aspect of the organism and accompanied by a generalized increase in mortality. Although a substantial number of candidates for biomarkers of aging have been proposed, none has been validated or universally accepted. Human telomeres constitute hexameric repetitive DNA sequence nucleoprotein complexes that cap chromosome ends, regulating gene expression and modulating stress-related pathways. Telomere length (TL) shortening is observed both in cellular senescence and advanced age, leading to the investigation of TL as a biomarker for aging and a risk factor indicator for the development and progression of the most common age-related diseases. The present review underlines the connection between TL and the pathophysiology of the diseases associated with telomere attrition. We performed a structured search of the PubMed database for peer-reviewed research of the literature regarding leukocyte TL and cardiovascular diseases (CVD), more specifically stroke and heart disease, and focused on the relevant articles published during the last 5 years. We also applied Hill's criteria of causation to strengthen this association. We analyzed the recent literature regarding TL length, stroke, and CVD. Although approximately one-third of the available studies support the connection, the results of different studies seem to be rather conflicting as a result of different study designs, divergent methods of TL determination, small study samples, and patient population heterogeneity. After applying Hill's criteria, we can observe that the literature conforms to them weakly, with chronology being the only Hill criterion of causality that probably cannot be contested. The present review attempted to examine the purported relation between leukocyte TL and age-related diseases such as CVD and more specific stroke and heart disease in view of the best established, comprehensive, medical and epidemiological criteria that have characterized the focused recent relevant research. Although several recommendations have been made that may contribute significantly to the field, a call for novel technical approaches and studies is mandatory to further elucidate the possible association.

Molecular biology of the cell

Molecular biology of the cell

RNA nucleoprotein complexes in biological systems

RNA-nucleoprotein (RNP) complexes are pivotal to the regulation of gene expression. These multiprotein, multifunctional complexes act as scaffolds, as molecular platforms, as hubs of interaction between the RNA and the proteins. The past decade has seen an exponential increase in the methodologies to identify and functionally characterize these complexes. In this review, we provide an overview of the recent advances in methodologies and the knowledge gained by characterizing these important complexes. This information has provided clues to the role of sequence, and structural determinants for RNA–Protein interaction networks in physiology and pathophysiology. With the recent studies implicating the role of RNA in the formation of biomolecular condensates, their role in health and disease warrants detailed sequence, structure and functional investigations.

Stochastically multimerized ParB orchestrates DNA assembly as unveiled by single-molecule analysis

The tripartite ParABS system mediates chromosome segregation in a wide range of bacteria. Dimeric ParB was proposed to nucleate on parS sites and spread to neighboring DNA. However, how properly distributed ParB dimers further compact chromosomal DNA into a higher-order nucleoprotein complex for partitioning remains poorly understood. Here, using a single-molecule approach, we show that tens of Bacillus subtilis ParB (Spo0J) proteins can stochastically multimerize on and stably bind to nonspecific DNA. The introduction of CTP promotes the formation and diffusion of the multimeric ParB along DNA, offering an opportunity for ParB proteins to further forgather and cluster. Intriguingly, ParB multimers can recognize parS motifs and are more inclined to remain immobile on them. Importantly, the ParB multimer features distinct capabilities of not only bridging two independent DNA molecules but also mediating their transportation, both of which are enhanced by the presence of either CTP or parS in the DNA. These findings shed new light on ParB dynamics in self-multimerization and DNA organization and help to better comprehend the assembly of the ParB-DNA partition complex.

Genus-Specific Interactions of Bacterial Chromosome Segregation Machinery Are Critical for Their Function.

Most bacteria use the ParABS system to segregate their newly replicated chromosomes. The two protein components of this system from various bacterial species share their biochemical properties: ParB is a CTPase that binds specific centromere-like parS sequences to assemble a nucleoprotein complex, while the ParA ATPase forms a dimer that binds DNA non-specifically and interacts with ParB complexes. The ParA-ParB interaction incites the movement of ParB complexes toward the opposite cell poles. However, apart from their function in chromosome segregation, both ParAB may engage in genus-specific interactions with other protein partners. One such example is the polar-growth controlling protein DivIVA in Actinomycetota, which binds ParA in Mycobacteria while interacts with ParB in Corynebacteria. Here, we used heterologous hosts to investigate whether the interactions between DivIVA and ParA or ParB are maintained across phylogenic classes. Specifically, we examined interactions of proteins from four bacterial species, two belonging to the Gram positive Actinomycetota phylum and two belonging to the Gram-negative Pseudomonadota. We show that while the interactions between ParA and ParB are preserved for closely related orthologs, the interactions with polarly localised protein partners are not conferred by orthologous ParABs. Moreover, we demonstrate that heterologous ParA cannot substitute for endogenous ParA, despite their high sequence similarity. Therefore, we conclude that ParA orthologs are fine-tuned to interact with their partners, especially their interactions with polarly localised proteins are adjusted to particular bacterial species demands.

Shelterin Components Modulate Nucleic Acids Condensation and Phase Separation in the Context of Telomeric DNA

Telomeres are nucleoprotein complexes that protect the ends of chromosomes and are essential for chromosome stability in Eukaryotes. In cells, individual telomeres form distinct globules of finite size that appear to be smaller than expected for bare DNA. Moreover, telomeres can cluster together, form telomere-induced-foci or co-localize with promyelocytic leukemia (PML) nuclear bodies. The physical basis for collapse of individual telomeres and coalescence of multiple ones remains unclear, as does the relationship between these two phenomena. By combining single-molecule force spectroscopy measurements, optical microscopy, turbidity assays, and simulations, we show that the telomere scaffolding protein TRF2 can condense individual DNA chains and drives coalescence of multiple DNA molecules, leading to phase separation and the formation of liquid-like droplets. Addition of the TRF2 binding protein hRap1 modulates phase boundaries and tunes the specificity of solution demixing while simultaneously altering the degree of DNA compaction. Our results suggest that the condensation of single telomeres and formation of biomolecular condensates containing multiple telomeres are two different outcomes driven by the same set of molecular interactions. Moreover, binding partners, such as other telomere components, can alter those interactions to promote single-chain DNA compaction over multiple-chain phase separation.

Imaging the Response to DNA Damage in Heterochromatin Domains.

The eukaryotic genome is assembled in a nucleoprotein complex called chromatin, whose organization markedly influences the repair of DNA lesions. For instance, compact chromatin states, broadly categorized as heterochromatin, present a challenging environment for DNA damage repair. Through transcriptional silencing, heterochromatin also plays a vital role in the maintenance of genomic integrity and cellular homeostasis. It is thus of critical importance to decipher whether and how heterochromatin affects the DNA damage response (DDR) to understand how this chromatin state is preserved after DNA damage. Here, we present two laser micro-irradiation-based methods for imaging the DDR in heterochromatin domains in mammalian cells. These methods allow DNA damage targeting to specific subnuclear compartments, direct visualization of the DDR and image-based quantification of the repair response. We apply them to study DNA double-strand break repair pathways in facultative heterochromatin and the repair of UV photoproducts in constitutive heterochromatin. We discuss the advantages and limitations of these methods compared to other targeted approaches for DNA damage induction.

Multivalent interactions essential for lentiviral integrase function

A multimer of retroviral integrase (IN) synapses viral DNA ends within a stable intasome nucleoprotein complex for integration into a host cell genome. Reconstitution of the intasome from the maedi-visna virus (MVV), an ovine lentivirus, revealed a large assembly containing sixteen IN subunits1. Herein, we report cryo-EM structures of the lentiviral intasome prior to engagement of target DNA and following strand transfer, refined at 3.4 and 3.5 Å resolution, respectively. The structures elucidate details of the protein-protein and protein-DNA interfaces involved in lentiviral intasome formation. We show that the homomeric interfaces involved in IN hexadecamer formation and the α-helical configuration of the linker connecting the C-terminal and catalytic core domains are critical for MVV IN strand transfer activity in vitro and for virus infectivity. Single-molecule microscopy in conjunction with photobleaching reveals that the MVV intasome can bind a variable number, up to sixteen molecules, of the lentivirus-specific host factor LEDGF/p75. Concordantly, ablation of endogenous LEDGF/p75 results in gross redistribution of MVV integration sites in human and ovine cells. Our data confirm the importance of the expanded architecture observed in cryo-EM studies of lentiviral intasomes and suggest that this organization underlies multivalent interactions with chromatin for integration targeting to active genes.