Dependence of nucleosome mechanical stability on DNA mismatches.

The organization of nucleosomes into chromatin and their accessibility are shaped by local DNA mechanics. Conversely, nucleosome positions shape genetic variations, which may originate from mismatches during replication and chemical modification of DNA. To investigate how DNA mismatches affect the mechanical stability and the exposure of nucleosomal DNA, we used an optical trap combined with single-molecule FRET and a single-molecule FRET cyclization assay. We found that a single base-pair C-C mismatch enhances DNA bendability and nucleosome mechanical stability for the 601-nucleosome positioning sequence. An increase in force required for DNA unwrapping from the histone core is observed for single base-pair C-C mismatches placed at three tested positions: at the inner turn, at the outer turn, or at the junction of the inner and outer turn of the nucleosome. The results support a model where nucleosomal DNA accessibility is reduced by mismatches, potentially explaining the preferred accumulation of single-nucleotide substitutions in the nucleosome core and serving as the source of genetic variation during evolution and cancer progression. Mechanical stability of an intact nucleosome, that is mismatch-free, is also dependent on the species as we find that yeast nucleosomes are mechanically less stable and more symmetrical in the outer turn unwrapping compared to Xenopus nucleosomes.

Determination of single-molecule loading rate during mechanotransduction in cell adhesion.

Cells connect with their environment through surface receptors and use physical tension in receptor-ligand bonds for various cellular processes. Single-molecule techniques have revealed bond strength by measuring "rupture force," but it has long been recognized that rupture force is dependent on loading rate-how quickly force is ramped up. Thus, the physiological loading rate needs to be measured to reveal the mechanical strength of individual bonds in their functional context. We have developed an overstretching tension sensor (OTS) to allow more accurate force measurement in physiological conditions with single-molecule detection sensitivity even in mechanically active regions. We used serially connected OTSs to show that the integrin loading rate ranged from 0.5 to 4 piconewtons per second and was about three times higher in leukocytes than in epithelial cells.

Free-energy measuring nanopore device.

Free energies (FEs) in molecular sciences can be used to quantify the stability of folded molecules. In this article, we introduce nanopores for measuring FEs. We pull DNA hairpin-forming molecules through a nanopore, measure work, and estimate the FE change in the slow limit, and with the Jarzynski fluctuation theorem (FT) at fast pulling times. We also pull our molecule with optical tweezers, compare it to nanopores, and explore how sampling single molecules from equilibrium or a folded ensemble affects the FE estimate via the FT. The nanopore experiment helps us address and overcome the conceptual problem of equilibrium sampling in single-molecule pulling experiments. Only when molecules are sampled from an equilibrium ensemble do nanopore and tweezer FE estimates mutually agree. We demonstrate that nanopores are very useful tools for comparing FEs of two molecules at finite times and we propose future applications.

Dynamic 1D search and processive nucleosome translocations by RSC and ISW2 chromatin remodelers.

Eukaryotic gene expression is linked to chromatin structure and nucleosome positioning by ATP-dependent chromatin remodelers that establish and maintain nucleosome-depleted regions (NDRs) near transcription start sites. Conserved yeast RSC and ISW2 remodelers exert antagonistic effects on nucleosomes flanking NDRs, but the temporal dynamics of remodeler search, engagement, and directional nucleosome mobilization for promoter accessibility are unknown. Using optical tweezers and two-color single-particle imaging, we investigated the Brownian diffusion of RSC and ISW2 on free DNA and sparse nucleosome arrays. RSC and ISW2 rapidly scan DNA by one-dimensional hopping and sliding, respectively, with dynamic collisions between remodelers followed by recoil or apparent co-diffusion. Static nucleosomes block remodeler diffusion resulting in remodeler recoil or sequestration. Remarkably, both RSC and ISW2 use ATP hydrolysis to translocate mono-nucleosomes processively at ~30 bp/s on extended linear DNA under tension. Processivity and opposing push-pull directionalities of nucleosome translocation shown by RSC and ISW2 shape the distinctive landscape of promoter chromatin.

Rapid Long-distance Migration of RPA on Single Stranded DNA Occurs Through Intersegmental Transfer Utilizing Multivalent Interactions.

Replication Protein A (RPA) is asingle strandedDNA(ssDNA)binding protein that coordinates diverse DNA metabolic processes including DNA replication, repair, and recombination. RPA is a heterotrimeric protein with six functional oligosaccharide/oligonucleotide (OB) domains and flexible linkers. Flexibility enables RPA to adopt multiple configurations andis thought to modulate its function. Here, usingsingle moleculeconfocal fluorescencemicroscopy combinedwith optical tweezers and coarse-grained molecular dynamics simulations, we investigated the diffusional migration of single RPA molecules on ssDNA undertension.The diffusioncoefficientDis the highest (20,000nucleotides2/s) at 3pNtension and in 100 mMKCl and markedly decreases whentensionor salt concentrationincreases. We attribute the tension effect to intersegmental transfer which is hindered by DNA stretching and the salt effect to an increase in binding site size and interaction energy of RPA-ssDNA. Our integrative study allowed us to estimate the size and frequency of intersegmental transfer events that occur through transient bridging of distant sites on DNA by multiple binding sites on RPA. Interestingly, deletion of RPA trimeric core still allowed significant ssDNA binding although the reduced contact area made RPA 15-fold more mobile. Finally, we characterized the effect of RPA crowding on RPA migration. These findings reveal how the high affinity RPA-ssDNA interactions are remodeled to yield access, a key step in several DNA metabolic processes.

Single-Macromolecule Studies of Eukaryotic Genomic Maintenance.

Genomes are self-organized and self-maintained as long, complex macromolecules of chromatin. The inherent heterogeneity, stochasticity, phase separation, and chromatin dynamics of genome operation make it challenging to study genomes using ensemble methods. Various single-molecule force-, fluorescent-, and sequencing-based techniques rooted in different disciplines have been developed to fill critical gaps in the capabilities of bulk measurements, each providing unique, otherwise inaccessible, insights into the structure and maintenance of the genome. Capable of capturing molecular-level details about the organization, conformational changes, and packaging of genetic material, as well as processive and stochastic movements of maintenance factors, a single-molecule toolbox provides an excellent opportunity for collaborative research to understand how genetic material functions in health and malfunctions in disease. In this review, we discuss novel insights brought to genomic sciences by single-molecule techniques and their potential to continue to revolutionize the field-one molecule at a time. Expected final online publication date for the Annual Review of Physical Chemistry, Volume 75 is April 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Dynamic 1D Search and Processive Nucleosome Translocations by RSC and ISW2 Chromatin Remodelers.

Eukaryotic gene expression is linked to chromatin structure and nucleosome positioning by ATP-dependent chromatin remodelers that establish and maintain nucleosome-depleted regions (NDRs) near transcription start-sites. Conserved yeast RSC and ISW2 remodelers exert antagonistic effects on nucleosomes flanking NDRs, but the temporal dynamics of remodeler search, engagement and directional nucleosome mobilization for promoter accessibility are unknown. Using optical tweezers and 2-color single-particle imaging, we investigated the Brownian diffusion of RSC and ISW2 on free DNA and sparse nucleosome arrays. RSC and ISW2 rapidly scan DNA by one-dimensional hopping and sliding respectively, with dynamic collisions between remodelers followed by recoil or apparent co-diffusion. Static nucleosomes block remodeler diffusion resulting in remodeler recoil or sequestration. Remarkably, both RSC and ISW2 use ATP hydrolysis to translocate mono-nucleosomes processively at ~30 bp/sec on extended linear DNA under tension. Processivity and opposing push-pull directionalities of nucleosome translocation shown by RSC and ISW2 shape the distinctive landscape of promoter chromatin.

Electrostatic encoding of genome organization principles within single native nucleosomes.

The eukaryotic genome, first packed into nucleosomes of about 150 bp around the histone core, is organized into euchromatin and heterochromatin, corresponding to the A and B compartments, respectively. Here, we asked if individual nucleosomes in vivo know where to go. That is, do mono-nucleosomes by themselves contain A/B compartment information, associated with transcription activity, in their biophysical properties? We purified native mono-nucleosomes to high monodispersity and used physiological concentrations of biological polyamines to determine their condensability. The chromosomal regions known to partition into A compartments have low condensability and vice versa. In silico chromatin polymer simulations using condensability as the only input showed that biophysical information needed to form compartments is all contained in single native nucleosomes and no other factors are needed. Condensability is also strongly anticorrelated with gene expression, and especially so near the promoter region and in a cell type dependent manner. Therefore, individual nucleosomes in the promoter know whether the gene is on or off, and that information is contained in their biophysical properties. Comparison with genetic and epigenetic features suggest that nucleosome condensability is a very meaningful axis onto which to project the high dimensional cellular chromatin state. Analysis of condensability using various condensing agents including those that are protein-based suggests that genome organization principle encoded into individual nucleosomes is electrostatic in nature. Polyamine depletion in mouse T cells, by either knocking out ornithine decarboxylase (ODC) or inhibiting ODC, results in hyperpolarized condensability, suggesting that when cells cannot rely on polyamines to translate biophysical properties of nucleosomes to control gene expression and 3D genome organization, they accentuate condensability contrast, which may explain dysfunction known to occur with polyamine deficiency.

SIKs Regulate HDAC7 Stabilization and Cytokine Recall in Late-Stage T Cell Effector Differentiation.

Understanding the mechanisms underlying the acquisition and maintenance of effector function during T cell differentiation is important to unraveling how these processes can be dysregulated in the context of disease and manipulated for therapeutic intervention. In this study, we report the identification of a previously unappreciated regulator of murine T cell differentiation through the evaluation of a previously unreported activity of the kinase inhibitor, BioE-1197. Specifically, we demonstrate that liver kinase B1 (LKB1)-mediated activation of salt-inducible kinases epigenetically regulates cytokine recall potential in effector CD8+ and Th1 cells. Evaluation of this phenotype revealed that salt-inducible kinase-mediated phosphorylation-dependent stabilization of histone deacetylase 7 (HDAC7) occurred during late-stage effector differentiation. HDAC7 stabilization increased nuclear HDAC7 levels, which correlated with total and cytokine loci-specific reductions in the activating transcription mark histone 3 lysine 27 acetylation (H3K27Ac). Accordingly, HDAC7 stabilization diminished transcriptional induction of cytokine genes upon restimulation. Inhibition of this pathway during differentiation produced effector T cells epigenetically poised for enhanced cytokine recall. This work identifies a previously unrecognized target for enhancing effector T cell functionality.

Enhanced mTORC1 signaling and protein synthesis in pathologic α-synuclein cellular and animal models of Parkinson's disease.

Pathologic α-synuclein plays an important role in the pathogenesis of α-synucleinopathies such as Parkinson's disease (PD). Disruption of proteostasis is thought to be central to pathologic α-synuclein toxicity; however, the molecular mechanism of this deregulation is poorly understood. Complementary proteomic approaches in cellular and animal models of PD were used to identify and characterize the pathologic α-synuclein interactome. We report that the highest biological processes that interacted with pathologic α-synuclein in mice included RNA processing and translation initiation. Regulation of catabolic processes that include autophagy were also identified. Pathologic α-synuclein was found to bind with the tuberous sclerosis protein 2 (TSC2) and to trigger the activation of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which augmented mRNA translation and protein synthesis, leading to neurodegeneration. Genetic and pharmacologic inhibition of mTOR and protein synthesis rescued the dopamine neuron loss, behavioral deficits, and aberrant biochemical signaling in the α-synuclein preformed fibril mouse model and Drosophila transgenic models of pathologic α-synuclein-induced degeneration. Pathologic α-synuclein furthermore led to a destabilization of the TSC1-TSC2 complex, which plays an important role in mTORC1 activity. Constitutive overexpression of TSC2 rescued motor deficits and neuropathology in α-synuclein flies. Biochemical examination of PD postmortem brain tissues also suggested deregulated mTORC1 signaling. These findings establish a connection between mRNA translation deregulation and mTORC1 pathway activation that is induced by pathologic α-synuclein in cellular and animal models of PD.

Directing Uphill Strand Displacement with an Engineered Superhelicase.

The ability to finely tune reaction rates and binding energies between components has made DNA strand displacement circuits promising candidates to replicate the complex regulatory functions of biological reaction networks. However, these circuits often lack crucial properties, such as signal turnover and the ability to transiently respond to successive input signals that require the continuous input of chemical energy. Here, we introduce a method for providing such energy to strand displacement networks in a controlled fashion: an engineered DNA helicase, Rep-X, that transiently dehybridizes specific DNA complexes, enabling the strands in the complex to participate in downstream hybridization or strand displacement reactions. We demonstrate how this process can direct the formation of specific metastable structures by design and that this dehybridization process can be controlled by DNA strand displacement reactions that effectively protect and deprotect a double-stranded complex from unwinding by Rep-X. These findings can guide the design of active DNA strand displacement regulatory networks, in which sustained dynamical behavior is fueled by helicase-regulated unwinding.

Mind your tag in single-molecule measurements.

In this issue of Cell Reports Methods, Molina and colleagues use in vitro single-molecule DNA flow-stretching to demonstrate the severe effects of appending a short lysine-cysteine-lysine (KCK) tag on the Bacillus subtilis ParB protein. This assay could be further utilized to evaluate the impact of other tags on DNA-binding proteins.

Dynamin 1xA interacts with Endophilin A1 via its spliced long C-terminus for ultrafast endocytosis.

Dynamin 1 (Dyn1) has two major splice variants, xA and xB, with unique C-terminal extensions of 20 and 7 amino acids, respectively. Of these, only Dyn1xA is enriched at endocytic zones and accelerates vesicle fission during ultrafast endocytosis. Here, we report that the long tail variant, Dyn1xA, achieves this localization by preferentially binding to Endophilin A through a newly defined Class II binding site overlapping with its extension, at a site spanning the splice boundary. Endophilin binds this site at higher affinity than the previously reported site, and this affinity is determined by amino acids outside the binding sites acting as long distance elements within the xA tail. Their interaction is regulated by the phosphorylation state of two serine residues specific to the xA variant. Dyn1xA and Endophilin colocalize in patches near the active zone of synapses. Mutations selectively disrupting Endophilin binding to the long extension cause Dyn1xA mislocalization along axons. In these mutants, endocytic pits are stalled on the plasma membrane during ultrafast endocytosis. These data suggest that the specificity for ultrafast endocytosis is defined by the phospho-regulated interaction of Endophilin A through a newly identified site of Dyn1xA's long tail.

Helicase Activity Modulation with On-Demand Light-Based Conformational Control.

Engineering a protein variant with a desired role relies on deep knowledge of the relationship between a protein's native structure and function. Using our structural understanding of a regulatory subdomain found in a family of DNA helicases, we engineered novel helicases for which the subdomain orientation is designed to switch between unwinding-inactive and -active conformations upon trans-cis isomerization of an azobenzene-based crosslinker. This on-demand light-based conformational control directly alters helicase activity as demonstrated by both bulk phase experiments and single-molecule optical tweezers analysis of one of the engineered helicases. The "opto-helicase" may be useful in future applications that require spatiotemporal control of DNA hybridization states.

Genome-wide analysis of DNA-PK-bound MRN cleavage products supports a sequential model of DSB repair pathway choice.

The Mre11-Rad50-Nbs1 (MRN) complex recognizes and processes DNA double-strand breaks for homologous recombination by performing short-range removal of 5' strands. Endonucleolytic processing by MRN requires a stably bound protein at the break site-a role we postulate is played by DNA-dependent protein kinase (DNA-PK) in mammals. Here we interrogate sites of MRN-dependent processing by identifying sites of CtIP association and by sequencing DNA-PK-bound DNA fragments that are products of MRN cleavage. These intermediates are generated most efficiently when DNA-PK is catalytically blocked, yielding products within 200 bp of the break site, whereas DNA-PK products in the absence of kinase inhibition show greater dispersal. Use of light-activated Cas9 to induce breaks facilitates temporal resolution of DNA-PK and Mre11 binding, showing that both complexes bind to DNA ends before release of DNA-PK-bound products. These results support a sequential model of double-strand break repair involving collaborative interactions between homologous and non-homologous repair complexes.

Bi-directional nucleosome sliding by the Chd1 chromatin remodeler integrates intrinsic sequence-dependent and ATP-dependent nucleosome positioning.

Chromatin remodelers use a helicase-type ATPase motor to shift DNA around the histone core. Although not directly reading out the DNA sequence, some chromatin remodelers exhibit a sequence-dependent bias in nucleosome positioning, which presumably reflects properties of the DNA duplex. Here, we show how nucleosome positioning by the Chd1 remodeler is influenced by local DNA perturbations throughout the nucleosome footprint. Using site-specific DNA cleavage coupled with next-generation sequencing, we show that nucleosomes shifted by Chd1 can preferentially localize DNA perturbations - poly(dA:dT) tracts, DNA mismatches, and single-nucleotide insertions - about a helical turn outside the Chd1 motor domain binding site, super helix location 2 (SHL2). This phenomenon occurs with both the Widom 601 positioning sequence and the natural +1 nucleosome sequence from the Saccharomyces cerevisiae SWH1 gene. Our modeling indicates that localization of DNA perturbations about a helical turn outward from SHL2 results from back-and-forth sliding due to remodeler action on both sides of the nucleosome. Our results also show that barrier effects from DNA perturbations can be extended by the strong phasing of nucleosome positioning sequences.

Linking folding dynamics and function of SAM/SAH riboswitches at the single molecule level.

Riboswitches are regulatory elements found in bacterial mRNAs that control downstream gene expression through ligand-induced conformational changes. Here, we used single-molecule FRET to map the conformational landscape of the translational SAM/SAH riboswitch and probe how co-transcriptional ligand-induced conformational changes affect its translation regulation function. Riboswitch folding is highly heterogeneous, suggesting a rugged conformational landscape that allows for sampling of the ligand-bound conformation even in the absence of ligand. The addition of ligand shifts the landscape, favoring the ligand-bound conformation. Mutation studies identified a key structural element, the pseudoknot helix, that is crucial for determining ligand-free conformations and their ligand responsiveness. We also investigated ribosomal binding site accessibility under two scenarios: pre-folding and co-transcriptional folding. The regulatory function of the SAM/SAH riboswitch involves kinetically favoring ligand binding, but co-transcriptional folding reduces this preference with a less compact initial conformation that exposes the Shine-Dalgarno sequence and takes min to redistribute to more compact conformations of the pre-folded riboswitch. Such slow equilibration decreases the effective ligand affinity. Overall, our study provides a deeper understanding of the complex folding process and how the riboswitch adapts its folding pattern in response to ligand, modulates ribosome accessibility and the role of co-transcriptional folding in these processes.

GAGA Factor Overcomes 1D Diffusion Barrier by 3D Diffusion in Search of Nucleosomal Targets.

The eukaryotic chromatin landscape plays important roles in DNA metabolism and is characterized by positioned nucleosomes near regulatory DNA, nucleosome-depleted regions and supranucleosomal organization. Nucleosome core histones limit DNA accessibility by structurally blocking half of the DNA surface and altering its topology, but how nucleosomes affect target search by sequence-specific transcription factors (TFs) remains enigmatic. Here, we used multi-color smFRET to investigate how Drosophila GAGA Factor (GAF) locates its targets. On free DNA, GAF rapidly diffuses in 1D to a single cognate motif but escapes after subsecond transient association. Nucleosomes effectively block 1D diffusion into its core, but GAF can bind, with surprisingly prolonged residence, at internal cognate sites by direct association from 3D. Our findings demonstrate the occlusive power of nucleosomes to 1D sliding and reveal that a combination of 1D and 3D diffusion by a zinc finger TF enables efficient target search on chromatin.

Bursting translation on single mRNAs in live cells.

Stochasticity has emerged as a mechanism of gene regulation. Much of this so-called "noise" has been attributed to bursting transcription. Although bursting transcription has been studied extensively, the role of stochasticity in translation has not been fully investigated due to the lack of enabling imaging technology. In this study, we developed techniques to track single mRNAs and their translation in live cells for hours, allowing the measurement of previously uncharacterized translation dynamics. We applied genetic and pharmacological perturbations to control translation kinetics and found that, like transcription, translation is not a constitutive process but instead cycles between inactive and active states, or "bursts." However, unlike transcription, which is largely frequency-modulated, complex structures in the 5'-untranslated region alter burst amplitudes. Bursting frequency can be controlled through cap-proximal sequences and trans-acting factors such as eIF4F. We coupled single-molecule imaging with stochastic modeling to quantitatively determine the kinetic parameters of translational bursting.

Membrane compression by synaptic vesicle exocytosis triggers ultrafast endocytosis.

Compensatory endocytosis keeps the membrane surface area of secretory cells constant following exocytosis. At chemical synapses, clathrin-independent ultrafast endocytosis maintains such homeostasis. This endocytic pathway is temporally and spatially coupled to exocytosis; it initiates within 50 ms at the region immediately next to the active zone where vesicles fuse. However, the coupling mechanism is unknown. Here, we demonstrate that filamentous actin is organized as a ring, surrounding the active zone at mouse hippocampal synapses. Assuming the membrane area conservation is due to this actin ring, our theoretical model suggests that flattening of fused vesicles exerts lateral compression in the plasma membrane, resulting in rapid formation of endocytic pits at the border between the active zone and the surrounding actin-enriched region. Consistent with model predictions, our data show that ultrafast endocytosis requires sufficient compression by exocytosis of multiple vesicles and does not initiate when actin organization is disrupted, either pharmacologically or by ablation of the actin-binding protein Epsin1. Our work suggests that membrane mechanics underlie the rapid coupling of exocytosis to endocytosis at synapses.