scholarly journals Single-molecule live cell imaging of Rep reveals the dynamic interplay between an accessory replicative helicase and the replisome

2019 ◽  
Vol 47 (12) ◽  
pp. 6287-6298 ◽  
Author(s):  
Aisha H Syeda ◽  
Adam J M Wollman ◽  
Alex L Hargreaves ◽  
Jamieson A L Howard ◽  
Jan-Gert Brüning ◽  
...  
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Atp Hydrolysis ◽  
Average Dwell Time ◽  
Genetic Studies ◽  
Replication Restart ◽  
Barrier Removal ◽  
Dynamic Interplay ◽  
Rapid Binding

Abstract DNA replication must cope with nucleoprotein barriers that impair efficient replisome translocation. Biochemical and genetic studies indicate accessory helicases play essential roles in replication in the presence of nucleoprotein barriers, but how they operate inside the cell is unclear. With high-speed single-molecule microscopy we observed genomically-encoded fluorescent constructs of the accessory helicase Rep and core replisome protein DnaQ in live Escherichia coli cells. We demonstrate that Rep colocalizes with 70% of replication forks, with a hexameric stoichiometry, indicating maximal occupancy of the single DnaB hexamer. Rep associates dynamically with the replisome with an average dwell time of 6.5 ms dependent on ATP hydrolysis, indicating rapid binding then translocation away from the fork. We also imaged PriC replication restart factor and observe Rep-replisome association is also dependent on PriC. Our findings suggest two Rep-replisome populations in vivo: one continually associating with DnaB then translocating away to aid nucleoprotein barrier removal ahead of the fork, another assisting PriC-dependent reloading of DnaB if replisome progression fails. These findings reveal how a single helicase at the replisome provides two independent ways of underpinning replication of protein-bound DNA, a problem all organisms face as they replicate their genomes.

scholarly journals DNA sliding and loop formation by E. coli SMC complex: MukBEF

2021 ◽  
Author(s):  
man zhou
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Dna Interaction ◽  
Loop Formation ◽  
Unknown Mechanism ◽  
E Coli ◽  
Dna Compaction ◽  
And Function ◽  
Structural Maintenance Of Chromosomes

SMC (structural maintenance of chromosomes) complexes share conserved architectures and function in chromosome maintenance via an unknown mechanism. Here we have used single-molecule techniques to study MukBEF, the SMC complex in Escherichia coli. Real-time movies show MukB alone can compact DNA and ATP inhibits DNA compaction by MukB. We observed that DNA unidirectionally slides through MukB, potentially by a ratchet mechanism, and the sliding speed depends on the elastic energy stored in the DNA. MukE, MukF and ATP binding stabilize MukB and DNA interaction, and ATP hydrolysis regulates the loading/unloading of MukBEF from DNA. Our data suggests a new model for how MukBEF organizes the bacterial chromosome in vivo; and this model will be relevant for other SMC proteins.

scholarly journals A molecularly engineered, broad-spectrum anti-coronavirus lectin inhibits SARS-CoV-2 and MERS-CoV infection in vivo

2021 ◽  
Author(s):  
David Markovitz ◽  
Jasper Chan ◽  
Yoo Jin Oh ◽  
Shuofeng Yuan ◽  
Hin Chu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Broad Spectrum ◽  
High Speed ◽  
Viral Infections ◽  
Healthy Human ◽  
Force Microscopy ◽  
Pulmonary Damage ◽  
High Mannose ◽  
Viral Components

Abstract Coronaviruses have repeatedly crossed species barriers to cause epidemics1. “Pan-coronavirus” antivirals targeting conserved viral components involved in coronavirus replication, such as the extensively glycosylated spike protein, can be designed. Here we show that the rationally engineered H84T-banana lectin (H84T-BanLec), which specifically recognizes high-mannose found on viral proteins but seldom on healthy human cells2, potently inhibits the highly virulent MERS-CoV, pandemic SARS-CoV-2 and its variants, and other human-pathogenic coronaviruses at nanomolar concentrations. MERS-CoV-infected human DPP4-transgenic mice treated by H84T-BanLec have significantly higher survival, lower viral burden, and reduced pulmonary damage. Similarly, prophylactic or therapeutic H84T-BanLec is effective against SARS-CoV-2 in hamsters. Importantly, intranasally and intraperitoneally administered H84T-BanLec are comparably effective. Time-of-drug-addition assay shows that H84T-BanLec targets virus entry. Real-time structural analysis with high-speed atomic force microscopy depicts multi-molecular associations of H84T-BanLec dimers with the SARS-CoV-2 spike trimer. Single-molecule force spectroscopy demonstrates binding of H84T-BanLec to multiple SARS-CoV-2 spike mannose sites with high affinity, and that H84T-BanLec competes with SARS-CoV-2 spike for binding to cellular ACE2. Modelling experiments identify distinct high-mannose glycans in spike recognized by H84T-BanLec. The multiple H84T-BanLec binding sites on spike likely account for the activity against SARS-CoV-2 variants and the lack of resistant mutants. The broad-spectrum H84T-BanLec should be clinically evaluated in respiratory viral infections including COVID-19.

scholarly journals Tracking DNA Replication Restart in vivo at the Single-Molecule Level

2020 ◽  
Vol 118 (3) ◽  
pp. 376a
Author(s):  
Alex L. Hargreaves ◽  
Aisha Syeda ◽  
Mark C. Leake
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Single Molecule Level ◽  
Replication Restart ◽  
Dna Replication Restart

scholarly journals Mobility of nucleosomes is regulated by their binding partners

2022 ◽  
Author(s):  
Daniel P Melters ◽  
Keir C Neuman ◽  
Tatini Rakshit ◽  
Yamini Dalal
Keyword(s):  
Atomic Force Microscopy ◽  
Single Molecule ◽  
High Speed ◽  
Diffusion Constant ◽  
Chromatin Accessibility ◽  
Chromatin Dynamics ◽  
Single Molecule Level ◽  
Force Microscopy ◽  
Atomic Force

Chromatin accessibility is modulated in a variety of ways, both to create open and closed chromatin states which are critical for eukaryotic gene regulation. At the mechanistic single molecule level, how accessibility is regulated remains a fundamental question in the field. Here, we use single molecule tracking by high-speed atomic force microscopy to investigate this question using chromatin arrays and extend our findings into the nucleus. By high-speed atomic force microscopy, we tracked chromatin dynamics in real time and observed that the essential kinetochore protein CENP-C reduces the diffusion constant of CENP-A nucleosomes and the linker H1.5 protein restricts H3 nucleosome mobility. We subsequently interrogated how CENP-C modulates CENP-A chromatin dynamics in vivo. Overexpressing CENP-C resulted in reduced centromeric transcription and impaired loading of new CENP-A molecules. These data suggest a model in which inner kinetochore proteins are critically involved in modulating chromatin accessibility and consequently, noncoding transcription at human centromeres.

scholarly journals A 2-dimensional ratchet model describes assembly initiation of a specialized bacterial cell surface

2019 ◽  
Vol 116 (43) ◽  
pp. 21789-21799 ◽  
Author(s):  
Emily A. Peluso ◽  
Taylor B. Updegrove ◽  
Jiji Chen ◽  
Hari Shroff ◽  
Kumaran S. Ramamurthi
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Bacterial Spores ◽  
Coat Proteins ◽  
Structural Protein ◽  
A Cell ◽  
Protein Shell ◽  
Ratchet Model

Bacterial spores are dormant cells that are encased in a thick protein shell, the “coat,” which participates in protecting the organism’s DNA from environmental insults. The coat is composed of dozens of proteins that assemble in an orchestrated fashion during sporulation. In Bacillus subtilis, 2 proteins initiate coat assembly: SpoVM, which preferentially binds to micron-scale convex membranes and marks the surface of the developing spore as the site for coat assembly; and SpoIVA, a structural protein recruited by SpoVM that uses ATP hydrolysis to drive its irreversible polymerization around the developing spore. Here, we describe the initiation of coat assembly by SpoVM and SpoIVA. Using single-molecule fluorescence microscopy in vivo in sporulating cells and in vitro on synthetic spores, we report that SpoVM’s localization is primarily driven by a lower off-rate on membranes of preferred curvature in the absence of other coat proteins. Recruitment and polymerization of SpoIVA results in the entrapment of SpoVM on the forespore surface. Using experimentally derived reaction parameters, we show that a 2-dimensional ratchet model can describe the interdependent localization dynamics of SpoVM and SpoIVA, wherein SpoVM displays a longer residence time on the forespore surface, which favors recruitment of SpoIVA to that location. Localized SpoIVA polymerization in turn prevents further sampling of other membranes by prelocalized SpoVM molecules. Our model therefore describes the dynamics of structural proteins as they localize and assemble at the correct place and time within a cell to form a supramolecular complex.

scholarly journals mRNA- and Factor-Driven Dynamic Variability Controls eIF4F-Cap Recognition for Translation Initiation.

2021 ◽  
Author(s):  
Burak Çetin ◽  
Seán E. O'Leary
Keyword(s):  
Single Molecule ◽  
Translational Control ◽  
Atp Hydrolysis ◽  
Translation Efficiency ◽  
Dependent Manner ◽  
Complex Interplay ◽  
Eif4f Complex ◽  
Mrna Length ◽  
Mrna Binding

mRNA 5′ cap recognition by eIF4F is a key step in eukaryotic translational control. While different mRNAs respond differently to eIF4F–directed regulation, the molecular basis for this variability remains unclear. We developed single-molecule fluorescence assays to directly observe eIF4F–mRNA interactions. We uncovered a complex interplay of mRNA features with factor activities that differentiates cap recognition between mRNAs. eIF4E–cap association rates are anticorrelated with mRNA length. eIF4A leverages ATP binding to differentially accelerate eIF4E–mRNA association; the extent of this acceleration correlates with translation efficiency in vivo. eIF4G lengthens eIF4E–cap binding to persist on the initiation timescale. The full eIF4F complex discriminates between mRNAs in an ATP-dependent manner. After eIF4F–mRNA binding, eIF4E is ejected from the cap by eIF4A ATP hydrolysis. Our results suggest features throughout mRNA coordinate in controlling cap recognition at the 5ʹ end, and suggest a model for how eIF4F–mRNA dynamics establish mRNA sensitivity to translational control processes.

scholarly journals Single-molecule live cell imaging of Rep reveals the dynamic interplay between an accessory replicative helicase and the replisome

2018 ◽  
Author(s):  
Aisha Syeda ◽  
Adam J. M. Wollman ◽  
Alex Hargreaves ◽  
Janny G. Brüning ◽  
Peter McGlynn ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
High Speed ◽  
Fluorescent Protein ◽  
Single Type ◽  
Replication Machinery ◽  
Replicative Helicase ◽  
Cellular Environment ◽  
New Findings

AbstractDNA replication requires strategies to cope with nucleoprotein barriers that impair the efficient translocation of the replisome. Biochemical and genetic studies indicate accessory helicases play essential roles in continuity of replication in the presence of nucleoprotein barriers, but how they operate in the native cellular environment is unclear. With high-speed single-molecule microscopy we determine the dynamic patterns of localization of genomically-encoded fluorescent protein constructs of the bacterial accessory helicase Rep and core replisome protein DnaQ in live E. coli cells. We demonstrate that Rep colocalizes with 70% of replication forks. Colocalisation is dependent upon interaction with replicative helicase DnaB, with an underlying hexameric stoichiometry of Rep indicating maximal occupancy of the single DnaB hexamer within the replisome. We find that Rep associates dynamically with the replisome with an average dwell time of 6.5 ms dependent on ATP hydrolysis, indicating rapid binding then translocation away from the fork. We also imaged the PriC replication restart factor given the known Rep-PriC functional interaction and observe Rep-replisome association is also dependent on the presence of PriC. Our findings suggest two Rep-replisome populations in vivo: one involving Rep continually associating with DnaB then translocating away to aid nucleoprotein barrier removal ahead of the fork, another assisting PriC-dependent reloading of DnaB if replisome progression fails. These new findings reveal how a single type of helicase is recruited to the replisome to provide two independent ways of underpinning replication of protein-bound DNA, a problem that all organisms face as they replicate their genomes.Significance statementAll organisms face the challenge of proteins bound to DNA acting as barriers to prevent DNA replication. We have performed fluorescence imaging experiments on living bacteria to track the positions of the replication machinery, a protein called Rep which is involved in removing these barriers, and a protein called PriC believed to be involved with reloading the replication machinery if the original replication machinery breaks down. We find that Rep is very dynamic with continual binding and movement away from the replication machinery. Association with the replication machinery depends on both binding to the replication machinery directly and on PriC. Thus Rep can circumvent barriers in two independent ways: a strategy which may be relevant to all organisms.

scholarly journals Single-Molecule Tracking of DNA Translocases inBacillus subtilisReveals Strikingly Different Dynamics of SftA, SpoIIIE, and FtsA

2018 ◽  
Vol 84 (8) ◽  
pp. e02610-17 ◽  
Author(s):  
Nina El Najjar ◽  
Jihad El Andari ◽  
Christine Kaimer ◽  
Georg Fritz ◽  
Thomas C. Rösch ◽  
...  
Keyword(s):  
Cell Division ◽  
Chromosome Segregation ◽  
Single Molecule ◽  
Average Dwell Time ◽  
Glycolytic Enzyme ◽  
Live Cells ◽  
Content Type ◽  
Dna Translocases ◽  
Membrane Bound

ABSTRACTLike many bacteria,Bacillus subtilispossesses two DNA translocases that affect chromosome segregation at different steps. Prior to septum closure, nonsegregated DNA is moved into opposite cell halves by SftA, while septum-entrapped DNA is rescued by SpoIIIE. We have used single-molecule fluorescence microscopy and tracking (SMT) experiments to describe the dynamics of the two different DNA translocases, the cell division protein FtsA and the glycolytic enzyme phosphofructokinase (PfkA), in real time. SMT revealed that about 30% of SftA molecules move through the cytosol, while a fraction of 70% is septum bound and static. In contrast, only 35% of FtsA molecules are static at midcell, while SpoIIIE molecules diffuse within the membrane and show no enrichment at the septum. Several lines of evidence suggest that FtsA plays a role in septal recruitment of SftA: anftsAdeletion results in a significant reduction in septal SftA recruitment and a decrease in the average dwell time of SftA molecules. FtsA can recruit SftA to the membrane in a heterologous eukaryotic system, suggesting that SftA may be partially recruited via FtsA. Therefore, SftA is a component of the division machinery, while SpoIIIE is not, and it is otherwise a freely diffusive cytosolic enzymein vivo. Our developed SMT script is a powerful technique to determine if low-abundance proteins are membrane bound or cytosolic, to detect differences in populations of complex-bound and unbound/diffusive proteins, and to visualize the subcellular localization of slow- and fast-moving molecules in live cells.IMPORTANCEDNA translocases couple the late events of chromosome segregation to cell division and thereby play an important role in the bacterial cell cycle. The proteins fall into one of two categories, integral membrane translocases or nonintegral translocases. We show that the membrane-bound translocase SpoIIIE moves slowly throughout the cell membrane inB. subtilisand does not show a clear association with the division septum, in agreement with the idea that it binds membrane-bound DNA, which can occur through cell division across nonsegregated chromosomes. In contrast, SftA behaves like a soluble protein and is recruited to the division septum as a component of the division machinery. We show that FtsA contributes to the recruitment of SftA, revealing a dual role of FtsA at the division machinery, but it is not the only factor that binds SftA. Our work represents a detailedin vivostudy of DNA translocases at the single-molecule level.

scholarly journals Rotary catalysis of bovine mitochondrial F1-ATPase studied by single-molecule experiments

2020 ◽  
Vol 117 (3) ◽  
pp. 1447-1456 ◽  
Author(s):  
Ryohei Kobayashi ◽  
Hiroshi Ueno ◽  
Chun-Biu Li ◽  
Hiroyuki Noji
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Atp Hydrolysis ◽  
Angular Position ◽  
Magnetic Tweezers ◽  
Reaction Scheme ◽  
Atp Binding ◽  
High Speed Imaging ◽  
Hydrolysis Process ◽  
Rotary Catalysis

The reaction scheme of rotary catalysis and the torque generation mechanism of bovine mitochondrial F1 (bMF1) were studied in single-molecule experiments. Under ATP-saturated concentrations, high-speed imaging of a single 40-nm gold bead attached to the γ subunit of bMF1 showed 2 types of intervening pauses during the rotation that were discriminated by short dwell and long dwell. Using ATPγS as a slowly hydrolyzing ATP derivative as well as using a functional mutant βE188D with slowed ATP hydrolysis, the 2 pausing events were distinctively identified. Buffer-exchange experiments with a nonhydrolyzable analog (AMP-PNP) revealed that the long dwell corresponds to the catalytic dwell, that is, the waiting state for hydrolysis, while it remains elusive which catalytic state short pause represents. The angular position of catalytic dwell was determined to be at +80° from the ATP-binding angle, mostly consistent with other F1s. The position of short dwell was found at 50 to 60° from catalytic dwell, that is, +10 to 20° from the ATP-binding angle. This is a distinct difference from human mitochondrial F1, which also shows intervening dwell that probably corresponds to the short dwell of bMF1, at +65° from the binding pause. Furthermore, we conducted “stall-and-release” experiments with magnetic tweezers to reveal how the binding affinity and hydrolysis equilibrium are modulated by the γ rotation. Similar to thermophilic F1, bMF1 showed a strong exponential increase in ATP affinity, while the hydrolysis equilibrium did not change significantly. This indicates that the ATP binding process generates larger torque than the hydrolysis process.

scholarly journals Single molecule mechanics resolves the earliest events in force generation by cardiac myosin

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Michael S Woody ◽  
Donald A Winkelmann ◽  
Marco Capitanio ◽  
E Michael Ostap ◽  
Yale E Goldman
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Atp Hydrolysis ◽  
Optical Trap ◽  
Force Generation ◽  
Actin Binding ◽  
Cardiac Myosin ◽  
Phosphate Release ◽  
Hydrolysis Products ◽  
Key Steps

Key steps of cardiac mechanochemistry, including the force-generating working stroke and the release of phosphate (Pi), occur rapidly after myosin-actin attachment. An ultra-high-speed optical trap enabled direct observation of the timing and amplitude of the working stroke, which can occur within <200 μs of actin binding by β-cardiac myosin. The initial actomyosin state can sustain loads of at least 4.5 pN and proceeds directly to the stroke or detaches before releasing ATP hydrolysis products. The rates of these processes depend on the force. The time between binding and stroke is unaffected by 10 mM Pi which, along with other findings, indicates the stroke precedes phosphate release. After Pi release, Pi can rebind enabling reversal of the working stroke. Detecting these rapid events under physiological loads provides definitive indication of the dynamics by which actomyosin converts biochemical energy into mechanical work.

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