Bulk Biochemical Assays Research Articles

HLTF disrupts Cas9-DNA post-cleavage complexes to allow DNA break processing

The outcome of CRISPR-Cas-mediated genome modifications is dependent on DNA double-strand break (DSB) processing and repair pathway choice. Homology-directed repair (HDR) of protein-blocked DSBs requires DNA end resection that is initiated by the endonuclease activity of the MRE11 complex. Using reconstituted reactions, we show that Cas9 breaks are unexpectedly not directly resectable by the MRE11 complex. In contrast, breaks catalyzed by Cas12a are readily processed. Cas9, unlike Cas12a, bridges the broken ends, preventing DSB detection and processing by MRE11. We demonstrate that Cas9 must be dislocated after DNA cleavage to allow DNA end resection and repair. Using single molecule and bulk biochemical assays, we next find that the HLTF translocase directly removes Cas9 from broken ends, which allows DSB processing by DNA end resection or non-homologous end-joining machineries. Mechanistically, the activity of HLTF requires its HIRAN domain and the release of the 3′-end generated by the cleavage of the non-target DNA strand by the Cas9 RuvC domain. Consequently, HLTF removes the H840A but not the D10A Cas9 nickase. The removal of Cas9 H840A by HLTF explains the different cellular impact of the two Cas9 nickase variants in human cells, with potential implications for gene editing.

Identical sequences, different behaviors: Protein diversity captured at the single-molecule level

The classical “one sequence, one structure, one function” paradigm has shaped much of our intuition of how proteins work inside the cell. Partially due to the insight provided by bulk biochemical assays, individual biomolecules are often assumed to behave as identical entities, and their characterization relies on ensemble averages that flatten any conformational diversity into a unique phenotype. While the emergence of single-molecule techniques opened the gates to interrogating individual molecules, technical shortcomings typically limit the duration of these measurements, which precludes a complete characterization of an individual protein and, hence, capturing the heterogeneity among molecular populations. Here, we introduce an ultrastable magnetic tweezers design, which enables us to measure the folding dynamics of a single protein during several uninterrupted days with high temporal and spatial resolution. Thanks to this instrumental development, we fully characterize the nanomechanics of two proteins with a very distinct force response, the talin R3IVVI domain and protein L. Days-long recordings on the same protein individual accumulate thousands of folding transitions with submicrosecond resolution, allowing us to reconstruct their free energy landscapes and describe how they evolve with force. By mapping the nanomechanical identity of many different protein individuals, we directly capture their molecular diversity as a quantifiable dispersion on their force response and folding kinetics. By significantly expanding the measurable timescales, our instrumental development offers a tool for profiling individual molecules, opening the gates to directly characterizing biomolecular heterogeneity.

RAD51 filament formation, dynamics, and regulation.

DNA damage can arise through various mechanisms such as exogenous sources (UV radiation) or endogenous sources (replication fork collapse). Damaged DNA poses a significant risk to the cell, as unrepaired damage can lead to mutations, gross chromosomal rearrangements, or even cell death. RAD51 is a highly conserved recombinase which serves crucial functions in the cell through its role in DNA damage repair (homologous recombination, HR) and in replication fork protection. In HR, RAD51 (with the help of mediators and paralogs) forms a nucleoprotein filament on the single-stranded DNA produced via resection of a double-stranded break. This nucleoprotein filament then invades the homologous sequence which will serve as a template for accurate repair of the damaged DNA. The mechanism by which the RAD51 nucleoprotein filament protects the replication fork is poorly understood. Slight deviations towards either a more or a less stable RAD51 filament may cause aberrant recombination, and/or failure to protect DNA replication forks by changing the filament assembly, stability and/or dynamics. Using a combination of single-molecule and bulk biochemical assays, this study examines filament dynamics of RAD51 in varying contexts: different physiological conditions, varying mutated residues, and with and without small molecule inhibitors known to alter RAD51 function. We use dual-optical tweezers combined with confocal microscopy to examine RAD51 filament formation in real time, with a microfluidic chip set up that allows for introduction of varying conditions for the same single DNA molecule. Following characterization of filament formation at the single-molecule level, DNA strand exchange assays are used to determine how variations on filament assembly and stability alter the primary recombinase function of RAD51. We show here that alterations to RAD51 filament formation, stability, or dynamics can alter the critical recombinase function of RAD51.

Studying the dynamics of human shelterin protein assembly on long telomeric DNA and their roles in telomerase recruitment using combined single-molecule manipulation and imaging

An important goal in the field of telomere biology is understanding the mechanism of telomere length regulation. Telomeres consist of long repetitive DNA sequences that are decorated by telomere-specific proteins. Shelterin, a major telomere protein complex, protects telomeres and regulates multiple steps of the telomerase telomere elongation pathway. Bulk biochemical assays have provided critical insights into how shelterin can both inhibit and stimulate telomerase catalytic activity. Still, these studies were performed using overly simplified and short telomeric DNA and did not reveal the complicated and dynamical processes that naturally come with long repetitive binding sites.

Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization.

Traditionally used for bulk biochemical assays, Xenopus laevis egg extracts have emerged as a powerful imaging-based tool for studying cytoplasmic phenomena, such as cytokinesis, mitotic spindle formation and assembly of the nucleus. Building upon early methods that imaged fixed extracts sampled at sparse time points, recent approaches image live extracts using time-lapse microscopy, revealing more dynamical features with enhanced temporal resolution. These methods usually require sophisticated surface treatments of the imaging vessel. Here we introduce an alternative method for live imaging of egg extracts that require no chemical surface treatment. It is simple to implement and utilizes mass-produced laboratory consumables for imaging. We describe a system that can be used for both wide-field and confocal microscopy. It is designed for imaging extracts in a 2-dimensional (2D) field, but can be easily extended to imaging in 3D. It is well-suited for studying spatial pattern formation within the cytoplasm. With representative data, we demonstrate the typical dynamic organization of microtubules, nuclei and mitochondria in interphase extracts prepared using this method. These image data can provide quantitative information on cytoplasmic dynamics and spatial organization.

Cohesin SA1 and SA2 are RNA binding proteins that localize to RNA containing regions on DNA.

Cohesin SA1 (STAG1) and SA2 (STAG2) are key components of the cohesin complex. Previous studies have highlighted the unique contributions by SA1 and SA2 to 3D chromatin organization, DNA replication fork progression, and DNA double-strand break (DSB) repair. Recently, we discovered that cohesin SA1 and SA2 are DNA binding proteins. Given the recently discovered link between SA2 and RNA-mediated biological pathways, we investigated whether or not SA1 and SA2 directly bind to RNA using a combination of bulk biochemical assays and single-molecule techniques, including atomic force microscopy (AFM) and the DNA tightrope assay. We discovered that both SA1 and SA2 bind to various RNA containing substrates, including ssRNA, dsRNA, RNA:DNA hybrids, and R-loops. Importantly, both SA1 and SA2 localize to regions on dsDNA that contain RNA. We directly compared the SA1/SA2 binding and R-loops sites extracted from Chromatin Immunoprecipitation sequencing (ChIP-seq) and DNA-RNA Immunoprecipitation sequencing (DRIP-Seq) data sets, respectively. This analysis revealed that SA1 and SA2 binding sites overlap significantly with R-loops. The majority of R-loop-localized SA1 and SA2 are also sites where other subunits of the cohesin complex bind. These results provide a new direction for future investigation of the diverse biological functions of SA1 and SA2.

A guide to visualizing the spatial epigenome with super-resolution microscopy.

Genomic DNA in eukaryotic cells is tightly compacted with histone proteins into nucleosomes, which are further packaged into the higher-order chromatin structure. The physical structuring of chromatin is highly dynamic and regulated by a large number of epigenetic modifications in response to various environmental exposures, both in normal development and pathological processes such as aging and cancer. Higher-order chromatin structure has been indirectly inferred by conventional bulk biochemical assays on cell populations, which do not allow direct visualization of the spatial information of epigenomics (referred to as spatial epigenomics). With recent advances in super-resolution microscopy, the higher-order chromatin structure can now be visualized invivo at an unprecedent resolution. This opens up new opportunities to study physical compaction of 3D chromatin structure in single cells, maintaining a well-preserved spatial context of tissue microenvironment. This review discusses the recent application of super-resolution fluorescence microscopy to investigate the higher-order chromatin structure of different epigenomic states. We also envision the synergistic integration of super-resolution microscopy and high-throughput genomic technologies for the analysis of spatial epigenomics to fully understand the genome function in normal biological processes and diseases.

Single-Molecule Studies on the Protein Translocon.

Single-molecule studies provide unprecedented details about processes that are difficult to grasp by bulk biochemical assays that yield ensemble-averaged results. One of these processes is the translocation and insertion of proteins across and into the bacterial cytoplasmic membrane. This process is facilitated by the universally conserved secretion (Sec) system, a multi-subunit membrane protein complex that consists of dissociable cytoplasmic targeting components, a molecular motor, a protein-conducting membrane pore, and accessory membrane proteins. Here, we review recent insights into the mechanisms of protein translocation and membrane protein insertion from single-molecule studies.

Mechanistic characterization of the DEAD-box RNA helicase Ded1 from yeast as revealed by a novel technique using single-molecule magnetic tweezers.

DEAD-box helicases are involved in all steps of RNA metabolism. They are ATP-dependent RNA binding proteins and RNA-dependent ATPases. They can displace short duplexes, but they lack processivity. Their mechanism and functioning are not clearly understood; classical or bulk biochemical assays are not sufficient to answer these questions. Single-molecule techniques provide useful tools, but they are limited in cases where the proteins are nonprocessive and give weak signals. We present here a new, magnetic-tweezers-based, single-molecule assay that is simple and that can sensitively measure the displacement time of a small, hybridized, RNA oligonucleotide. Tens of molecules can be analyzed at the same time. Comparing the displacement times with and without a helicase gives insights into the enzymatic activity of the protein. We used this assay to study yeast Ded1, which is orthologous to human DDX3. Although Ded1 acts on a variety of substrates, we find that Ded1 requires an RNA substrate for its ATP-dependent unwinding activity and that ATP hydrolysis is needed to see this activity. Further, we find that only intramolecular single-stranded RNA extensions enhance this activity. We propose a model where ATP-bound Ded1 stabilizes partially unwound duplexes and where multiple binding events may be needed to see displacement.

Single-Molecule Movies of the Interplay between DNA Polymerase and Single Strand DNA Binding (SSB) Protein

Reliable DNA replication is completed by the orchestrated activation of a multi-protein machinery, the replisome, in a highly coordinated and dynamic fashion. The bacteriophage T7 gene 2.5 protein (gp2.5) works as a single strand DNA(ssDNA) binding protein and has been demonstrated to play a key role in the maintenance of ssDNA stability as well as the suppression of hairpin formation during each cycle of Okazaki fragment synthesis. After gp2.5 binding to ssDNA, gp2.5 might act as a road block for DNA polymerase to proceed. Bulk biochemical assays, however, demonstrate that the presence of gp2.5 increases the processivity and rate of template extension, while a mutant gp2.5 without c-terminus decreases the activity. Here we present movies of the single-molecule dynamics of T7 DNA polymerase and gp2.5 using optical trapping combined with confocal fluorescence microscopy. First, we investigated at single molecule level the effect of presence of wide type and mutant gp2.5 on DNA polymerase activity under various conditions. Then, we observed in real-time the labelled gp2.5 binding and unbinding dynamics interacting with DNA polymerase. It demonstrates that gp2.5 binds with ssDNA at low force (<30pN) but that its binding to ssDNA decreases significantly at higher force (>30pN). Beside, fluorescence data shows labelled DNA polymerase appearing at multiple places on the DNA which indicates the DNA polymerase might bind to DNA at multiple sites. These results combined with further visualization of the two labeled proteins will help us better understand the interplay between DNA polymerase and gp2.5.

Single-Cell, Time-Lapse Reactive Oxygen Species Detection in E. coli.

Detection of reactive oxygen species (ROS) in bacteria has been limited to bulk biochemical assays. Although they are powerful and quantitative tools to understand the overall production of ROS in E. coli, such assays provide limited spatial and temporal information when correlating cellular phenotype with perturbations such as antibiotics or other treatments. We have developed single-cell, time-lapse assays to detect ROS in live E. coli. The assays utilize flow systems on a fluorescence microscope to correlate symptoms aroused from biological or chemical perturbations with the in situ detection of ROS. ROS is detected by fluorogenic dyes that accumulate inside the cell, allowing detection of ROS in single cells in both homogeneous and heterogeneous samples using CellROX Green and Amplex® Red/APEX2. © 2018 by John Wiley & Sons, Inc.

Single-Stranded DNA Curtains for Studying the Srs2 Helicase Using Total Internal Reflection Fluorescence Microscopy.

Helicases are crucial participants in many types of DNA repair reactions, including homologous recombination. The properties of these enzymes can be assayed by traditional bulk biochemical analysis; however, these types of assays cannot directly access some types of information. In particular, bulk biochemical assays cannot readily access information that may be obscured in population averages. Single-molecule assays offer the potential advantage of being able to visualize the molecules in question in real time, thus providing direct access to questions relating to translocation velocity, processivity, and insights into how helicases may behave on different types of substrates. Here, we describe the use of single-stranded DNA (ssDNA) curtains as an assay for directly viewing the behavior of the Saccharomyces cerevisiae Srs2 helicase on single molecules of ssDNA. When used with total internal reflection fluorescence microscopy, these methods can be used to track the binding and movements of individual helicase complexes, and allow new insights into helicase behaviors at the single-molecule level.

Single-molecule mechanochemical characterization of E. coli pol III core catalytic activity.

Pol III core is the three‐subunit subassembly of the E. coli replicative DNA polymerase III holoenzyme. It contains the catalytic polymerase subunit α, the 3′ → 5′ proofreading exonuclease ε, and a subunit of unknown function, θ. We employ optical tweezers to characterize pol III core activity on a single DNA substrate. We observe polymerization at applied template forces F < 25 pN and exonucleolysis at F > 30 pN. Both polymerization and exonucleolysis occur as a series of short bursts separated by pauses. For polymerization, the initiation rate after pausing is independent of force. In contrast, the exonucleolysis initiation rate depends strongly on force. The measured force and concentration dependence of exonucleolysis initiation fits well to a two‐step reaction scheme in which pol III core binds bimolecularly to the primer‐template junction, then converts at rate k 2 into an exo‐competent conformation. Fits to the force dependence of k init show that exo initiation requires fluctuational opening of two base pairs, in agreement with temperature‐ and mismatch‐dependent bulk biochemical assays. Taken together, our results support a model in which the pol and exo activities of pol III core are effectively independent, and in which recognition of the 3′ end of the primer by either α or ε is governed by the primer stability. Thus, binding to an unstable primer is the primary mechanism for mismatch recognition during proofreading, rather than an alternative model of duplex defect recognition.

Reconstitution of Protein Dynamics Involved in Bacterial Cell Division.

Even simple cells like bacteria have precisely regulated cellular anatomies, which allow them to grow, divide and to respond to internal or external cues with high fidelity. How spatial and temporal intracellular organization in prokaryotic cells is achieved and maintained on the basis of locally interacting proteins still remains largely a mystery. Bulk biochemical assays with purified components and in vivo experiments help us to approach key cellular processes from two opposite ends, in terms of minimal and maximal complexity. However, to understand how cellular phenomena emerge, that are more than the sum of their parts, we have to assemble cellular subsystems step by step from the bottom up. Here, we review recent in vitro reconstitution experiments with proteins of the bacterial cell division machinery and illustrate how they help to shed light on fundamental cellular mechanisms that constitute spatiotemporal order and regulate cell division.

Recruitment, Duplex Unwinding and Protein-Mediated Inhibition of the Dead-Box RNA Helicase Dbp2 at Actively Transcribed Chromatin

RNA helicases play fundamental roles in modulating RNA structures and facilitating RNA–protein (RNP) complex assembly in vivo. Previously, our laboratory demonstrated that the DEAD-box RNA helicase Dbp2 in Saccharomyces cerevisiae is required to promote efficient assembly of the co-transcriptionally associated mRNA-binding proteins Yra1, Nab2, and Mex67 onto poly(A)+RNA. We also found that Yra1 associates directly with Dbp2 and functions as an inhibitor of Dbp2-dependent duplex unwinding, suggestive of a cycle of unwinding and inhibition by Dbp2. To test this, we undertook a series of experiments to shed light on the order of events for Dbp2 in co-transcriptional mRNP assembly. We now show that Dbp2 is recruited to chromatin via RNA and forms a large, RNA-dependent complex with Yra1 and Mex67. Moreover, single-molecule fluorescence resonance energy transfer and bulk biochemical assays show that Yra1 inhibits unwinding in a concentration-dependent manner by preventing the association of Dbp2 with single-stranded RNA. This inhibition prevents over-accumulation of Dbp2 on mRNA and stabilization of a subset of RNA polymerase II transcripts. We propose a model whereby Yra1 terminates a cycle of mRNP assembly by Dbp2.

Rational design of DNA-actuated enzyme nanoreactors guided by single molecule analysis.

The control of enzymatic reactions using nanoscale DNA devices offers a powerful application of DNA nanotechnology uniquely derived from actuation. However, previous characterization of enzymatic reaction rates using bulk biochemical assays reported suboptimal function of DNA devices such as tweezers. To gain mechanistic insight into this deficiency and to identify design rules to improve their function, here we exploit the synergy of single molecule imaging and computational modeling to characterize the three-dimensional structures and catalytic functions of DNA tweezer-actuated nanoreactors. Our analysis revealed two important deficiencies--incomplete closure upon actuation and conformational heterogeneity. Upon rational redesign of the Holliday junctions located at their hinge and arms, we found that the DNA tweezers could be more completely and uniformly closed. A novel single molecule enzyme assay was developed to demonstrate that our design improvements yield significant, independent enhancements in the fraction of active enzyme nanoreactors and their individual substrate turnover frequencies. The sequence-level design strategies explored here may aid more broadly in improving the performance of DNA-based nanodevices including biological and chemical sensors.

Real-Time Observation of DNA Recognition by the RNA-Guided Endonuclease Cas9 using Single-Molecule FRET

The RNA-guided endonuclease Cas9 targets foreign DNA for degradation as part of an adaptive immune system in bacteria mediated by clustered regularly interspaced short palindromic repeats (CRISPR). Due in part to the ease of programmability, Cas9 is now being widely used in various organisms for site-specific genomic editing, genome-wide knockout screens, and transcriptional activation and repression. This system brings together three major classes of biopolymers (DNA, RNA and protein), but the specificity of different components for each other remains poorly understood. Perhaps most important, Cas9 has been shown to bind and cleave genomic DNA sequences with varying degrees of mismatches to the guide RNA, leading to potentially deleterious off-target effects. Previous methods to investigate the tolerability of mismatches have largely employed experimental methods that use Cas9-induced DNA cleavage as a readout of specificity and not direct binding. Recent ChIP-seq experiments suggest that off-target binding occurs at sites with complementarity in just a short 5-bp seed region, but the use of crosslinking obscures kinetic information and can potentially introduce artifacts. Here, we have used single-molecule FRET together with bulk biochemical assays to directly observe interactions between Cas9-RNA and DNA targets of varying sequence complementarity in real time. Using multiple distinct labeling geometries that report on both initial association and subsequent DNA unwinding, we develop a kinetic framework for the RNA-guided DNA interrogation process and provide insight into conformational changes that are required for target cleavage. Our findings resolve outstanding questions on the mechanism of DNA recognition by Cas9 and will facilitate improvements in genome engineering applications.

DNA-Intercalation Kinetics Elucidated by Single-Dye Fluorescence Microscopy and Force Spectroscopy

Intercalating dyes are versatile molecules for fluorescence visualization of double-stranded DNA. Despite their widespread use in single-molecule and bulk biochemical assays, a complete mechanistic and kinetic insight in their interaction with DNA is lacking, which hampers optimization of biological assays using these molecules. Here, we study intercalation of double-stranded DNA by visualizing single DNA-intercalation events in real-time using fluorescence microscopy and controlling DNA tension with optical tweezers. This allows, for the first time, to fully quantify the affinity and kinetics of the mono-intercalators SYTOX Orange, SYTOX Green, SYBR Gold, and YO-PRO-1, and of the bis-intercalators YOYO-1, and POPO-3. We show that all these intercalators obey single-step, first-order DNA-binding kinetics and that their affinity for DNA is governed by an off-rate that decreases exponentially with DNA tension. Remarkably, the DNA tension alters the DNA-intercalator interaction time by two to four orders of magnitude. In addition to this strong tension dependence, the affinity for DNA also depends strongly on the intercalator species and on the ionic strength of the buffer. These new fundamental insights provide opportunities to selectively perform experiments either in or far out-of-equilibrium. We demonstrate that this kinetic control can be exploited to optimize biological assays based on intercalation: it allows minimizing the perturbation that intercalators impose on the dynamics of structural transitions during DNA overstretching and on processive enzymatic reactions such as DNA replication.

Enterococcus faecalis Produces Abundant Extracellular Structures Containing DNA in the Absence of Cell Lysis during Early Biofilm Formation

ABSTRACTEnterococcus faecalis is a common Gram-positive commensal bacterium of the metazoan gastrointestinal tract capable of biofilm formation and an opportunistic pathogen of increasing clinical concern. Dogma has held that biofilms are slow-growing structures, often taking days to form mature microcolonies. Here we report that extracellular DNA (eDNA) is an integral structural component of early E. faecalis biofilms (≤4 h postinoculation). Combining cationic dye-based biofilm matrix stabilization techniques with correlative immuno-scanning electron microscopy (SEM) and fluorescent techniques, we demonstrate that—in early E. faecalis biofilms—eDNA localizes to previously undescribed intercellular filamentous structures, as well as to thick mats of extruded extracellular matrix material. Both of these results are consistent with previous reports that early biofilms are exquisitely sensitive to exogenous DNase treatment. High-resolution SEM demonstrates a punctate labeling pattern in both structures, suggesting the presence of an additional, non-DNA constituent. Notably, the previously described fratricidal or lytic mechanism reported as the source of eDNA in older (≥24 h) E. faecalis biofilms does not appear to be at work under these conditions; extensive visual examination by SEM revealed a striking lack of lysed cells, and bulk biochemical assays also support an absence of significant lysis at these early time points. In addition, some cells demonstrated eDNA labeling localized at the septum, suggesting the possibility of DNA secretion from metabolically active cells. Overall, these data are consistent with a model in which a subpopulation of viable E. faecalis cells secrete or extrude DNA into the extracellular matrix.

Single Molecule Analysis of the Arabidopsis FRA1 Kinesin Shows that It Is a Functional Motor Protein with Unusually High Processivity

The Arabidopsis FRA1 kinesin contributes to the organization of cellulose microfibrils through an unknown mechanism. The cortical localization of this kinesin during interphase raises the possibility that it transports cell wall-related cargoes along cortical microtubules that either directly or indirectly influence cellulose microfibril patterning. To determine whether FRA1 is an authentic motor protein, we combined bulk biochemical assays and single molecule fluorescence imaging to analyze the motor properties of recombinant, GFP-tagged FRA1 containing the motor and coiled-coil domains (designated as FRA1(707)-GFP). We found that FRA1(707)-GFP binds to microtubules in an ATP-dependent manner and that its ATPase activity is dramatically stimulated by the presence of microtubules. Using single molecule studies, we found that FRA1(707)-GFP moves processively along microtubule tracks at a velocity of about 0.4 μm s(-1). In addition, we found that FRA1(707)-GFP is a microtubule plus-end-directed motor and that it moves along microtubules as a dimer. Interestingly, our single molecule analysis shows that the processivity of FRA1(707)-GFP is at least twice the processivity of conventional kinesin, making FRA1 the most processive kinesin to date. Together, our data show that FRA1 is a bona fide motor protein that has the potential to drive long-distance transport of cargo along cortical microtubules.