Antoine van Oijen

Endocytosis by Random Initiation and Stabilization of Clathrin-Coated Pits

Publisher: Elsevier BV

Date: 09-2004

DOI: 10.1016/J.CELL.2004.08.017

Single-particle kinetics of influenza virus membrane fusion

Publisher: Proceedings of the National Academy of Sciences

Date: 07-10-2008

DOI: 10.1073/PNAS.0807771105

Abstract: Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays are generally limited to observation of ensembles of multiple fusion events, confounding more detailed analysis of the sequence of the molecular steps involved. We have developed an in vitro , two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support. Analysis of lipid- and content-mixing trajectories on a particle-by-particle basis provides evidence for multiple, long-lived kinetic intermediates leading to hemifusion, followed by a single, rate-limiting step to pore formation. We interpret the series of intermediates preceding hemifusion as a result of the requirement that multiple copies of the trimeric hemagglutinin fusion protein be activated to initiate the fusion process.

Unraveling the electronic structure of individual photosynthetic pigment-protein complexes

Publisher: American Association for the Advancement of Science (AAAS)

Date: 16-07-1999

DOI: 10.1126/SCIENCE.285.5426.400

Abstract: Low-temperature single-molecule spectroscopic techniques were applied to a light-harvesting pigment-protein complex (LH2) from purple photosynthetic bacteria. The properties of the electronically excited states of the two circular assemblies (B800 and B850) of bacteriochlorophyll a (BChl a) pigment molecules in the in idual complexes were revealed, without ensemble averaging. The results show that the excited states of the B800 ring of pigments are mainly localized on in idual BChl a molecules. In contrast, the absorption of a photon by the B850 ring can be consistently described in terms of an excitation that is completely delocalized over the ring. This property may contribute to the high efficiency of energy transfer in these photosynthetic complexes.

Shining a Spotlight on DNA: Single-Molecule Methods to Visualise DNA

Publisher: MDPI AG

Date: 30-01-2019

DOI: 10.3390/MOLECULES24030491

Abstract: The ability to watch single molecules of DNA has revolutionised how we study biological transactions concerning nucleic acids. Many strategies have been developed to manipulate DNA molecules to investigate mechanical properties, dynamics and protein–DNA interactions. Imaging methods using small molecules and protein-based probes to visualise DNA have propelled our understanding of complex biochemical reactions involving DNA. This review focuses on summarising some of the methodological developments made to visualise in idual DNA molecules and discusses how these probes have been used in single-molecule biophysical assays.

A base-excision DNA-repair protein finds intrahelical lesion bases by fast sliding in contact with DNA

Publisher: Proceedings of the National Academy of Sciences

Date: 11-04-2006

DOI: 10.1073/PNAS.0509723103

Abstract: A central mystery in the function of site-specific DNA-binding proteins is the detailed mechanism for rapid location and binding of target sites in DNA. Human oxoguanine DNA glycosylase 1 (hOgg1), for ex le, must search out rare 8-oxoguanine lesions to prevent transversion mutations arising from oxidative stress. Here we report high-speed imaging of single hOgg1 enzyme molecules diffusing along DNA stretched by shear flow. Salt-concentration-dependent measurements reveal that such diffusion occurs as hOgg1 slides in persistent contact with DNA. At near-physiologic pH and salt concentration, hOgg1 has a subsecond DNA-binding time and slides with a diffusion constant as high as 5 × 10 6 bp 2 /s. Such a value approaches the theoretical upper limit for one-dimensional diffusion and indicates an activation barrier for sliding of only 0.5 kcal/mol (1 kcal = 4.2 kJ). This nearly barrierless Brownian sliding indicates that DNA glycosylases locate lesion bases by a massively redundant search in which the enzyme selectively binds 8-oxoguanine under kinetic control.

Spatial and temporal organization of RecA in the Escherichia coli DNA-damage response

Publisher: eLife Sciences Publications, Ltd

Date: 05-02-2019

DOI: 10.7554/ELIFE.42761

Abstract: The RecA protein orchestrates the cellular response to DNA damage via its multiple roles in the bacterial SOS response. Lack of tools that provide unambiguous access to the various RecA states within the cell have prevented understanding of the spatial and temporal changes in RecA structure/function that underlie control of the damage response. Here, we develop a monomeric C-terminal fragment of the λ repressor as a novel fluorescent probe that specifically interacts with RecA filaments on single-stranded DNA (RecA*). Single-molecule imaging techniques in live cells demonstrate that RecA is largely sequestered in storage structures during normal metabolism. Upon DNA damage, the storage structures dissolve and the cytosolic pool of RecA rapidly nucleates to form early SOS-signaling complexes, maturing into DNA-bound RecA bundles at later time points. Both before and after SOS induction, RecA* largely appears at locations distal from replisomes. Upon completion of repair, RecA storage structures reform.

CHD4 slides nucleosomes by decoupling entry- and exit-side DNA translocation.

Publisher: Springer Science and Business Media LLC

Date: 23-03-2020

DOI: 10.1038/S41467-020-15183-2

Abstract: Chromatin remodellers hydrolyse ATP to move nucleosomal DNA against histone octamers. The mechanism, however, is only partially resolved, and it is unclear if it is conserved among the four remodeller families. Here we use single-molecule assays to examine the mechanism of action of CHD4, which is part of the least well understood family. We demonstrate that the binding energy for CHD4-nucleosome complex formation—even in the absence of nucleotide—triggers significant conformational changes in DNA at the entry side, effectively priming the system for remodelling. During remodelling, flanking DNA enters the nucleosome in a continuous, gradual manner but exits in concerted 4–6 base-pair steps. This decoupling of entry- and exit-side translocation suggests that ATP-driven movement of entry-side DNA builds up strain inside the nucleosome that is subsequently released at the exit side by DNA expulsion. Based on our work and previous studies, we propose a mechanism for nucleosome sliding.

Tunability of DNA polymerase stability during eukaryotic DNA replication

Publisher: Cold Spring Harbor Laboratory

Date: 08-04-2019

DOI: 10.1101/602086

Abstract: Structural and biochemical studies have revealed the basic principles of how the replisome duplicates genomic DNA, but little is known about its dynamics during DNA replication. We reconstitute the 34 proteins needed to form the S. cerevisiae replisome and show how changing local concentrations of the key DNA polymerases tunes the ability of the complex to efficiently recycle these proteins or to dynamically exchange them. Particularly, we demonstrate redundancy of the Pol α DNA polymerase activity in replication and show that Pol α primase and the lagging-strand Pol δ can be re-used within the replisome to support the synthesis of large numbers of Okazaki fragments. This unexpected malleability of the replisome might allow it to deal with barriers and resource challenges during replication of large genomes.

Simultaneous single-molecule measurements of phage T7 replisome composition and function reveal the mechanism of polymerase exchange

Publisher: Proceedings of the National Academy of Sciences

Date: 18-01-2011

DOI: 10.1073/PNAS.1018824108

Abstract: A complete understanding of the molecular mechanisms underlying the functioning of large, multiprotein complexes requires experimental tools capable of simultaneously visualizing molecular architecture and enzymatic activity in real time. We developed a novel single-molecule assay that combines the flow-stretching of in idual DNA molecules to measure the activity of the DNA-replication machinery with the visualization of fluorescently labeled DNA polymerases at the replication fork. By correlating polymerase stoichiometry with DNA synthesis of T7 bacteriophage replisomes, we are able to quantitatively describe the mechanism of polymerase exchange. We find that even at relatively modest polymerase concentration (∼2 nM), soluble polymerases are recruited to an actively synthesizing replisome, dramatically increasing local polymerase concentration. These excess polymerases remain passively associated with the replisome through electrostatic interactions with the T7 helicase for ∼50 s until a stochastic and transient dissociation of the synthesizing polymerase from the primer-template allows for a polymerase exchange event to occur.

Steric exclusion and protein conformation determine the localization of plasma membrane transporters

Publisher: Springer Science and Business Media LLC

Date: 05-02-2018

DOI: 10.1038/S41467-018-02864-2

Abstract: The plasma membrane (PM) of Saccharomyces cerevisiae contains membrane compartments, MCC/eisosomes and MCPs, named after the protein residents Can1 and Pma1, respectively. Using high-resolution fluorescence microscopy techniques we show that Can1 and the homologous transporter Lyp1 are able to diffuse into the MCC/eisosomes, where a limited number of proteins are conditionally trapped at the (outer) edge of the compartment. Upon addition of substrate, the immobilized proteins diffuse away from the MCC/eisosomes, presumably after taking a different conformation in the substrate-bound state. Our data indicate that the mobile fraction of all integral plasma membrane proteins tested shows extremely slow Brownian diffusion through most of the PM. We also show that proteins with large cytoplasmic domains, such as Pma1 and synthetic chimera of Can1 and Lyp1, are excluded from the MCC/eisosomes. We hypothesize that the distinct localization patterns found for these integral membrane proteins in S. cerevisiae arises from a combination of slow lateral diffusion, steric exclusion, and conditional trapping in membrane compartments.

On the Spatial Organization of mRNA, Plasmids, and Ribosomes in a Bacterial Host Overexpressing Membrane Proteins

Publisher: Public Library of Science (PLoS)

Date: 15-12-2016

DOI: 10.1371/JOURNAL.PGEN.1006523

Sequence-dependent sliding kinetics of p53

Publisher: Proceedings of the National Academy of Sciences

Date: 25-09-2012

DOI: 10.1073/PNAS.1120452109

Abstract: Proper timing of gene expression requires that transcription factors (TFs) efficiently locate and bind their target sites within a genome. Theoretical studies have long proposed that one-dimensional sliding along DNA while simultaneously reading its sequence can accelerate TF’s location of target sites. Sliding by prokaryotic and eukaryotic TFs were subsequently observed. More recent theoretical investigations have argued that simultaneous reading and sliding is not possible for TFs without their possessing at least two DNA-binding modes. The tumor suppressor p53 has been shown to slide on DNA, and recent experiments have offered structural and single molecule support for a two-mode model for the protein. If the model is applicable to p53, then the requirement that TFs be able to read while sliding implies that noncognate sites will affect p53’s mobility on DNA, which will thus be generally sequence-dependent. Here, we confirm this prediction with single-molecule microscopy measurements of p53’s local diffusivity on noncognate DNA. We show how a two-mode model accurately predicts the variation in local diffusivity, while a single-mode model does not. We further determine that the best model of sequence-specific binding energy includes terms for “hemi-specific” binding, with one dimer of tetrameric p53 binding specifically to a half-site and the other binding nonspecifically to noncognate DNA. Our work provides evidence that the recognition by p53 of its targets and the timing thereof can depend on its noncognate binding properties and its ability to change between multiple modes of binding, in addition to the much better-studied effects of cognate-site binding.

Single-Molecule Fluorescence Studies of Membrane Transporters Using Total Internal Reflection Microscopy

Publisher: Elsevier

Date: 2017

DOI: 10.1016/BS.MIE.2017.05.009

Abstract: Cells are delineated by a lipid bilayer that physically separates the inside from the outer environment. Most polar, charged, or large molecules require proteins to reduce the energetic barrier for passage across the membrane and to achieve transport rates that are relevant for life. Here, we describe techniques to visualize the functioning of membrane transport proteins with fluorescent probes at the single-molecule level. First, we explain how to produce membrane-reconstituted transporters with fluorescent labels. Next, we detail the construction of a microfluidic flow cell to image immobilized proteoliposomes on a total internal reflection fluorescence microscope. We conclude by describing the methods that are needed to analyze fluorescence movies and obtain useful single-molecule data.

A single molecule DNA flow stretching microscope for undergraduates

Publisher: American Association of Physics Teachers (AAPT)

Date: 18-10-2011

DOI: 10.1119/1.3620410

Abstract: The design of a simple, safe, and inexpensive single molecule flow stretching instrument is presented. The instrument uses a low cost upright microscope coupled to a webcam for imaging single DNA molecules that are tethered in an easy to construct microfluidic flow cell. The system requires no special vibration isolation and is capable of measuring DNA replication at the single molecule level. We discuss two laboratory experiments suitable for advanced undergraduates using our microscope.

Single-Molecule Observation of Prokaryotic DNA Replication

Publisher: Springer New York

Date: 2015

DOI: 10.1007/978-1-4939-2596-4_14

Abstract: Replication of DNA requires the coordinated activity of a number of proteins within a multiprotein complex, the replisome. Recent advances in single-molecule techniques have enabled the observation of dynamic behavior of in idual replisome components and of the replisome as a whole, aspects that previously often have been obscured by ensemble averaging in more classical solution-phase biochemical experiments. To improve robustness and reproducibility of single-molecule assays of replication and allow objective analysis and comparison of results obtained from such assays, common practices should be established. Here, we describe the technical details of two assays to study replisome activity. In one, the kinetics of replication are observed as length changes in DNA molecules mechanically stretched by a laminar flow applied to attached beads. In the other, fluorescence imaging is used to determine both the kinetics and stoichiometry of in idual replisome components. These in vitro single-molecule methods allow for elucidation of the dynamic behavior of in idual replication proteins of prokaryotic replication systems.

The role of auxiliary domains in modulating CHD4 activity suggests mechanistic commonality between enzyme families

Publisher: Springer Science and Business Media LLC

Date: 06-12-2022

DOI: 10.1038/S41467-022-35002-0

Abstract: CHD4 is an essential, widely conserved ATP-dependent translocase that is also a broad tumour dependency. In common with other SF2-family chromatin remodelling enzymes, it alters chromatin accessibility by repositioning histone octamers. Besides the helicase and adjacent tandem chromodomains and PHD domains, CHD4 features 1000 residues of N- and C-terminal sequence with unknown structure and function. We demonstrate that these regions regulate CHD4 activity through different mechanisms. An N-terminal intrinsically disordered region (IDR) promotes remodelling integrity in a manner that depends on the composition but not sequence of the IDR. The C-terminal region harbours an auto-inhibitory region that contacts the helicase domain. Auto-inhibition is relieved by a previously unrecognized C-terminal SANT-SLIDE domain split by ~150 residues of disordered sequence, most likely by binding of this domain to substrate DNA. Our data shed light on CHD4 regulation and reveal strong mechanistic commonality between CHD family members, as well as with ISWI-family remodellers.

Hemagglutinin-Mediated Membrane Fusion: A Biophysical Perspective

Publisher: Annual Reviews

Date: 20-05-2018

DOI: 10.1146/ANNUREV-BIOPHYS-070317-033018

Abstract: Influenza hemagglutinin (HA) is a viral membrane protein responsible for the initial steps of the entry of influenza virus into the host cell. It mediates binding of the virus particle to the host-cell membrane and catalyzes fusion of the viral membrane with that of the host. HA is therefore a major target in the development of antiviral strategies. The fusion of two membranes involves high activation barriers and proceeds through several intermediate states. Here, we provide a biophysical description of the membrane fusion process, relating its kinetic and thermodynamic properties to the large conformational changes taking place in HA and placing these in the context of multiple HA proteins working together to mediate fusion. Furthermore, we highlight the role of novel single-particle experiments and computational approaches in understanding the fusion process and their complementarity with other biophysical approaches.

Mechanism of membrane fusion induced by vesicular stomatitis virus G protein

Publisher: Proceedings of the National Academy of Sciences

Date: 14-12-2017

DOI: 10.1073/PNAS.1618883114

Abstract: Enveloped viruses—those with a lipid-bilayer membrane such as influenza, dengue, and human immunodeficiency viruses—enter cells by fusion of the viral membrane with a membrane of the cell. A viral surface glycoprotein, known as its “fusion protein,” facilitates this step. Previous work studying the kinetics of single virus particles fusing with a target membrane has outlined a mechanism by which conformational changes in the fusion protein accelerate merger of the two bilayers. In this paper, we extend that mechanism to a structurally distinct class of viral fusion proteins, providing strong evidence for its general applicability to all viral membrane fusion processes.

Single-particle fusion of influenza viruses reveals complex interactions with target membranes

Publisher: IOP Publishing

Date: 27-04-2018

DOI: 10.1088/1361-648X/AABC21

Real-time single-molecule observation of chaperone-assisted protein folding

Publisher: Cold Spring Harbor Laboratory

Date: 10-05-2022

DOI: 10.1101/2022.05.09.491114

Abstract: The ability of Hsp70 molecular chaperones to remodel the conformation of their clients is central to their biological function however, questions remain regarding the precise molecular mechanisms by which Hsp70 machinery interacts with the client and how this contributes towards efficient protein folding. Here, we used Total Internal Reflection Fluorescence (TIRF) microscopy and single-molecule Fluorescence Resonance Energy Transfer (smFRET) to temporally observe the conformational changes that occur to in idual firefly luciferase (Fluc) proteins as they are folded by the bacterial Hsp70 system. For the first time, we observed multiple cycles of chaperone binding-and-release to an in idual client during refolding and that high rates of chaperone cycling improves refolding yield. Furthermore, we demonstrate that DnaJ remodels misfolded proteins via a conformational selection mechanism whereas DnaK resolves misfolded states via mechanical unfolding. This study illustrates that the temporal observation of chaperone-assisted folding enables the elucidation of key mechanistic details inaccessible using other approaches.

Mutations for Worse or Better: Low-Fidelity DNA Synthesis by SOS DNA Polymerase V Is a Tightly Regulated Double-Edged Sword

Publisher: American Chemical Society (ACS)

Date: 12-04-2016

DOI: 10.1021/ACS.BIOCHEM.6B00117

Single-molecule approaches to characterizing kinetics of biomolecular interactions

Publisher: Elsevier BV

Date: 02-2011

DOI: 10.1016/J.COPBIO.2010.10.002

Abstract: Single-molecule fluorescence techniques have emerged as powerful tools to study biological processes at the molecular level. This review describes the application of these methods to the characterization of the kinetics of interaction between biomolecules. A large number of single-molecule assays have been developed that visualize association and dissociation kinetics in vitro by fluorescently labeling binding partners and observing their interactions over time. Even though recent progress has been significant, there are certain limitations to this approach. To allow the observation of in idual, fluorescently labeled molecules requires low, nanomolar concentrations. I will discuss how such concentration requirements in single-molecule experiments limit their applicability to investigate intermolecular interactions and how recent technical advances deal with this issue.

Frequent template switching in postreplication gaps: suppression of deleterious consequences by the Escherichia coli Uup and RadD proteins

Publisher: Oxford University Press (OUP)

Date: 30-10-2020

DOI: 10.1093/NAR/GKZ960

Abstract: When replication forks encounter template DNA lesions, the lesion is simply skipped in some cases. The resulting lesion-containing gap must be converted to duplex DNA to permit repair. Some gap filling occurs via template switching, a process that generates recombination-like branched DNA intermediates. The Escherichia coli Uup and RadD proteins function in different pathways to process the branched intermediates. Uup is a UvrA-like ABC family ATPase. RadD is a RecQ-like SF2 family ATPase. Loss of both functions uncovers frequent and RecA-independent deletion events in a plasmid-based assay. Elevated levels of crossing over and repeat expansions accompany these deletion events, indicating that many, if not most, of these events are associated with template switching in postreplication gaps as opposed to simple replication slippage. The deletion data underpin simulations indicating that multiple postreplication gaps may be generated per replication cycle. Both Uup and RadD bind to branched DNAs in vitro. RadD protein suppresses crossovers and Uup prevents nucleoid mis-segregation. Loss of Uup and RadD function increases sensitivity to ciprofloxacin. We present Uup and RadD as genomic guardians. These proteins govern two pathways for resolution of branched DNA intermediates such that potentially deleterious genome rearrangements arising from frequent template switching are averted.

Erratum: Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited

Publisher: Springer Science and Business Media LLC

Date: 03-2006

DOI: 10.1038/NCHEMBIO0306-168

Nitrifier‐induced denitrification is an important source of soil nitrous oxide and can be inhibited by a nitrification inhibitor 3,4‐dimethylpyrazole phosphate

Publisher: Wiley

Date: 15-08-2017

DOI: 10.1111/1462-2920.13872

Abstract: Soil ecosystem represents the largest contributor to global nitrous oxide (N

Design of customizable long linear DNA substrates with controlled end modifications for single-molecule studies

Publisher: Elsevier BV

Date: 03-2020

DOI: 10.1016/J.AB.2019.113541

Abstract: Many strategies have been developed to manipulate DNA molecules and investigate protein-DNA interactions with single-molecule resolution. Often, these require long DNA molecules with a length of several 10s of kb that are chemically modified at specific regions. This need has traditionally been met by commercially available DNA from bacteriophage λ. However, λ DNA does not allow for the generation of highly customizable substrates in a straightforward manner, an important factor when developing assays to study complex biochemical reactions. Here we present a generalizable method for the design and production of very long chemically modified DNA substrates derived from a single plasmid. We show the versatility of this design by demonstrating its application in studying DNA replication in vitro. We anticipate this strategy will be broadly useful in producing a range of long chemically modified DNA molecules required for a erse range of single-molecule approaches.

Stability versus exchange: a paradox in DNA replication

Publisher: Oxford University Press (OUP)

Date: 25-04-2016

DOI: 10.1093/NAR/GKW296

E. coli DNA replication in the absence of free β clamps

Publisher: Wiley

Date: 25-03-2011

DOI: 10.1038/EMBOJ.2011.84

A versatile approach to the generation of fluid supported lipid bilayers and its applications

Publisher: Wiley

Date: 28-05-2014

DOI: 10.1002/BIT.25273

Abstract: Establishing supported lipid bilayers with biologically relevant composition, including transmembrane proteins and various classes of lipids, presents a significant challenge. We describe a generic and facile approach to the production of fluid polymer-supported lipid bilayers that allows for the incorporation of a wide variety of lipids and transmembrane proteins. The method is based on the formation of a polymer brush displaying lipid groups, followed by spin-coating of membrane lipids. Subsequentially, transmembrane proteins are incorporated by the fusion of proteoliposomes with the bilayer. Several applications, including the incorporation and single-molecule tracking of transmembrane proteins in a bilayer and the visualization of the fusion of in idual, membrane-enveloped viruses with a supported membrane, are demonstrated. Our results suggest that the membrane properties are consistent with those found in physiologically relevant conditions and underscore the wide applicability of our approach to synthetic biology, lab-on-a-chip applications, biophysical and pharmaceutical studies.

CLASP Promotes Microtubule Rescue by Recruiting Tubulin Dimers to the Microtubule

Publisher: Elsevier BV

Date: 08-2010

DOI: 10.1016/J.DEVCEL.2010.07.016

Tumor suppressor p53 slides on DNA with low friction and high stability

Publisher: Elsevier BV

Date: 07-2008

DOI: 10.1529/BIOPHYSJ.108.134122

Resolving the Gordian Knot: Srs2 Strips Intermediates Formed during Homologous Recombination

Publisher: Elsevier BV

Date: 03-2018

DOI: 10.1016/J.TIBS.2017.12.004

Abstract: Cells use a suite of specialized enzymes to repair chromosomal double-strand breaks (DSBs). Two recent studies describe how single-molecule fluorescence imaging techniques are used in the direct visualization of some of the key molecular steps involved. De Tullio et al. and Kaniecki et al. watch in idual Srs2 helicase molecules disrupt repair intermediates formed by RPA, Rad51, and Rad52 on DNA during homologous recombination.

Single-molecule analysis of DNA replication in Xenopus egg extracts

Publisher: Elsevier BV

Date: 06-2012

DOI: 10.1016/J.YMETH.2012.03.033

3-Dimensional super-resolution by spectrally selective imaging

Publisher: Elsevier BV

Date: 07-1998

DOI: 10.1016/S0009-2614(98)00673-3

The small heat shock protein Hsp27 binds α-synuclein fibrils, preventing elongation and cytotoxicity

Publisher: Elsevier BV

Date: 03-2018

DOI: 10.1074/JBC.M117.813865

Spectroscopy on the B850 Band of Individual Light-Harvesting 2 Complexes of Rhodopseudomonas acidophila II. Exciton States of an Elliptically Deformed Ring Aggregate

Publisher: Elsevier BV

Date: 03-2001

DOI: 10.1016/S0006-3495(01)76133-4

Special Issue on Cell Biophysics

Publisher: Elsevier BV

Date: 07-2018

DOI: 10.1016/J.BPJ.2018.06.017

Direct observation of enzymes replicating DNA using a single-molecule DNA stretching assay

Publisher: MyJove Corporation

Date: 23-03-2010

DOI: 10.3791/1689

Dynamic DNA Helicase-DNA Polymerase Interactions Assure Processive Replication Fork Movement

Publisher: Elsevier BV

Date: 08-2007

DOI: 10.1016/J.MOLCEL.2007.06.020

Abstract: A single copy of bacteriophage T7 DNA polymerase and DNA helicase advance the replication fork with a processivity greater than 17,000 nucleotides. Nonetheless, the polymerase transiently dissociates from the DNA without leaving the replisome. Ensemble and single-molecule techniques demonstrate that this dynamic processivity is made possible by two modes of DNA polymerase-helicase interaction. During DNA synthesis the polymerase and the helicase interact at a high-affinity site. In this polymerizing mode, the polymerase dissociates from the DNA approximately every 5000 bases. The polymerase, however, remains bound to the helicase via an electrostatic binding mode that involves the acidic C-terminal tail of the helicase and a basic region in the polymerase to which the processivity factor also binds. The polymerase transfers via the electrostatic interaction around the hexameric helicase in search of the primer-template.

Role of RNase H enzymes in maintaining genome stability in Escherichia coli expressing a steric-gate mutant of pol VICE391

Publisher: Elsevier BV

Date: 12-2019

DOI: 10.1016/J.DNAREP.2019.102685

Single-molecule studies of complex systems: the replisome

Publisher: Royal Society of Chemistry (RSC)

Date: 2007

DOI: 10.1039/B612545J

Abstract: A complete, system-level understanding of biological processes requires comprehensive information on the kinetics and thermodynamics of the underlying biochemical reactions. A wide variety of structural, biochemical, and molecular biological techniques have led to a quantitative understanding of the molecular properties and mechanisms essential to the processes of life. Yet, the ensemble averaging inherent to these techniques limits us in understanding the dynamic behavior of the molecular participants. Recent advances in imaging and molecular manipulation techniques have made it possible to observe the activity of in idual enzymes and record "molecular movies" that provide insight into their dynamics and reaction mechanisms. An important future goal is extending the applicability of single-molecule techniques to the study of larger, more complex multi-protein systems. In this review, the DNA replication machinery will be used as an ex le to illustrate recent progress in the development of various single-molecule techniques and its contribution to our understanding of the orchestration of multiple enzymatic processes in large biomolecular systems.

DNA polymerase IV primarily operates outside of DNA replication forks in Escherichia coli

Publisher: Public Library of Science (PLoS)

Date: 19-01-2018

DOI: 10.1371/JOURNAL.PGEN.1007161

Single-molecule visualization of stalled replication-fork rescue by theEscherichia coliRep helicase

Publisher: Cold Spring Harbor Laboratory

Date: 05-12-2022

DOI: 10.1101/2022.12.04.519054

Abstract: Genome duplication occurs while the template DNA is bound by numerous DNA-binding proteins. Each of these proteins act as potential roadblocks to the replication fork and can have deleterious effects on cells. In Escherichia coli , these roadblocks are displaced by the accessory helicase Rep, a DNA translocase and helicase that interacts with the replisome. The mechanistic details underlying the coordination with replication and roadblock removal by Rep remain poorly understood. Through real-time fluorescence imaging of the DNA produced by in idual E. coli replisomes and the simultaneous visualization of fluorescently-labeled Rep, we show that Rep continually surveils elongating replisomes. We found that this association of Rep with the replisome is stochastic and occurs independently of whether the fork is stalled or not. Further, we visualize the efficient rescue of stalled replication forks by directly imaging in idual Rep molecules as they remove a model protein roadblock, dCas9, from the template DNA. Using roadblocks of varying DNA-binding stabilities, we conclude that replication restart is the rate-limiting step of stalled replication rescue.

DNA primase acts as a molecular brake in DNA replication

Publisher: Springer Science and Business Media LLC

Date: 02-2006

DOI: 10.1038/NATURE04317

Abstract: A hallmark feature of DNA replication is the coordination between the continuous polymerization of nucleotides on the leading strand and the discontinuous synthesis of DNA on the lagging strand. This synchronization requires a precisely timed series of enzymatic steps that control the synthesis of an RNA primer, the recycling of the lagging-strand DNA polymerase, and the production of an Okazaki fragment. Primases synthesize RNA primers at a rate that is orders of magnitude lower than the rate of DNA synthesis by the DNA polymerases at the fork. Furthermore, the recycling of the lagging-strand DNA polymerase from a finished Okazaki fragment to a new primer is inherently slower than the rate of nucleotide polymerization. Different models have been put forward to explain how these slow enzymatic steps can take place at the lagging strand without losing coordination with the continuous and fast leading-strand synthesis. Nonetheless, a clear picture remains elusive. Here we use single-molecule techniques to study the kinetics of a multiprotein replication complex from bacteriophage T7 and to characterize the effect of primase activity on fork progression. We observe the synthesis of primers on the lagging strand to cause transient pausing of the highly processive leading-strand synthesis. In the presence of both leading- and lagging-strand synthesis, we observe the formation and release of a replication loop on the lagging strand. Before loop formation, the primase acts as a molecular brake and transiently halts progression of the replication fork. This observation suggests a mechanism that prevents leading-strand synthesis from outpacing lagging-strand synthesis during the slow enzymatic steps on the lagging strand.

Simultaneous Real-Time Imaging of Leading and Lagging Strand Synthesis Reveals the Coordination Dynamics of Single Replisomes

Publisher: Elsevier BV

Date: 12-2016

DOI: 10.1016/J.MOLCEL.2016.10.028

Abstract: The molecular machinery responsible for DNA replication, the replisome, must efficiently coordinate DNA unwinding with priming and synthesis to complete duplication of both strands. Due to the anti-parallel nature of DNA, the leading strand is copied continuously, while the lagging strand is produced by repeated cycles of priming, DNA looping, and Okazaki-fragment synthesis. Here, we report a multidimensional single-molecule approach to visualize this coordination in the bacteriophage T7 replisome by simultaneously monitoring the kinetics of loop growth and leading-strand synthesis. We show that loops in the lagging strand predominantly occur during priming and only infrequently support subsequent Okazaki-fragment synthesis. Fluorescence imaging reveals polymerases remaining bound to the lagging strand behind the replication fork, consistent with Okazaki-fragment synthesis behind and independent of the replication complex. In idual replisomes display both looping and pausing during priming, reconciling ergent models for the regulation of primer synthesis and revealing an underlying plasticity in replisome operation.

Tunability of DNA Polymerase Stability during Eukaryotic DNA Replication

Publisher: Elsevier BV

Date: 2020

DOI: 10.1016/J.MOLCEL.2019.10.005

Dancing on DNA: Kinetic Aspects of Search Processes on DNA

Publisher: Wiley

Date: 10-05-2011

DOI: 10.1002/CPHC.201100112

Single-molecule studies of polymerase dynamics and stoichiometry at the bacteriophage T7 replication machinery

Publisher: Proceedings of the National Academy of Sciences

Date: 03-03-2014

DOI: 10.1073/PNAS.1402010111

Abstract: The DNA replication machinery is a multiprotein assembly that is responsible for a rapid and faithful duplication of the genome. Based on biochemical, structural, and genetic studies, we have obtained a very detailed picture of its different enzymatic activities that underlie unwinding of the parental duplex DNA and incorporation of nucleotides into daughter DNA. However, little is known about the dynamic changes in composition of the complex during replication. Here, we use single-molecule fluorescence methods to visualize the dynamics with which in idual DNA polymerases, the replication protein responsible for DNA synthesis, associate with and dissociate from the replication machinery. Our results show that the DNA polymerase remains associated with the replication complex for a much shorter time than our previous models suggested.

The innate immune sensor IFI16 recognizes foreign DNA in the nucleus by scanning along the duplex

Publisher: eLife Sciences Publications, Ltd

Date: 16-12-2015

DOI: 10.7554/ELIFE.11721

Abstract: The ability to recognize foreign double-stranded (ds)DNA of pathogenic origin in the intracellular environment is an essential defense mechanism of the human innate immune system. However, the molecular mechanisms underlying distinction between foreign DNA and host genomic material inside the nucleus are not understood. By combining biochemical assays and single-molecule techniques, we show that the nuclear innate immune sensor IFI16 one-dimensionally tracks long stretches of exposed foreign dsDNA to assemble into supramolecular signaling platforms. We also demonstrate that nucleosomes represent barriers that prevent IFI16 from targeting host DNA by directly interfering with these one-dimensional movements. This unique scanning-assisted assembly mechanism allows IFI16 to distinguish friend from foe and assemble into oligomers efficiently and selectively on foreign DNA.

Bypass of a protein barrier by a replicative DNA helicase

Publisher: Springer Science and Business Media LLC

Date: 28-11-2012

DOI: 10.1038/NATURE11730

Spotting the Mistakes, One Molecule at a Time

Publisher: Elsevier BV

Date: 07-2012

DOI: 10.1016/J.STR.2012.06.007

Abstract: In this issue of Structure, Cho and colleagues provide intriguing insight into the first steps of the DNA mismatch repair process. By using single-molecule techniques, they show that the protein MutS undergoes two different types of diffusion on error-containing DNA in an ATP-dependent way.

Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis

Publisher: Springer Science and Business Media LLC

Date: 23-11-2009

DOI: 10.1038/NATURE07512

Single-Molecule Imaging at High Fluorophore Concentrations by Local Activation of Dye

Publisher: Elsevier BV

Date: 02-2015

DOI: 10.1016/J.BPJ.2014.12.019

Cutting the forest to see a single tree?

Publisher: Springer Science and Business Media LLC

Date: 08-2008

DOI: 10.1038/NCHEMBIO0808-440

Abstract: The development of single-molecule tools has significantly impacted the way we think about biochemical processes. Watching a single protein in action allows us to observe kinetic details and rare subpopulations that are hidden in ensemble-averaging techniques. I will discuss here the pros and cons of the single-molecule approach in studying ligand binding in macromolecular systems and how these techniques can be applied to characterize the behavior of large multicomponent biochemical systems.

Replication-Fork Dynamics

Publisher: Cold Spring Harbor Laboratory

Date: 23-07-2013

DOI: 10.1101/CSHPERSPECT.A010157

Single-molecule imaging reveals molecular coupling between transcription and DNA repair machinery in live cells

Publisher: Springer Science and Business Media LLC

Date: 20-03-2020

DOI: 10.1038/S41467-020-15182-3

Abstract: The Escherichia coli transcription-repair coupling factor Mfd displaces stalled RNA polymerase and delivers the stall site to the nucleotide excision repair factors UvrAB for damage detection. Whether this handoff from RNA polymerase to UvrA occurs via the Mfd-UvrA 2 -UvrB complex or alternate reaction intermediates in cells remains unclear. Here, we visualise Mfd in actively growing cells and determine the catalytic requirements for faithful recruitment of nucleotide excision repair proteins. We find that ATP hydrolysis by UvrA governs formation and disassembly of the Mfd-UvrA 2 complex. Further, Mfd-UvrA 2 -UvrB complexes formed by UvrB mutants deficient in DNA loading and damage recognition are impaired in successful handoff. Our single-molecule dissection of interactions of Mfd with its partner proteins inside live cells shows that the dissociation of Mfd is tightly coupled to successful loading of UvrB, providing a mechanism via which loading of UvrB occurs in a strand-specific manner.

Replisome bypass of a protein-based R-loop block by Pif1

Publisher: Proceedings of the National Academy of Sciences

Date: 16-11-2020

DOI: 10.1073/PNAS.2020189117

Abstract: The replisome machine that duplicates the DNA genome encounters a variety of blocks to replication, such as DNA bound proteins, R-loops, and DNA lesions. Studies in bacterial systems demonstrate that replisome advance through blocks is facilitated by an accessory monomeric helicase that acts on the opposite strand from the replicative hexameric helicase. This report examines this issue in a eukaryotic system, using Pif1, a monomeric helicase that acts on the opposite strand from the replicative CMG helicase. The report shows that an inactive “dead” Cas9 (dCas9) protein R-loop block arrests the replisome, but Pif1 enables replisome bypass of the dCas9 R-loop block. Hence, use of monomeric helicases may have evolved to aid replisome bypass of protein-DNA and protein-bound R-loop blocks.

Thioredoxin suppresses microscopic hopping of T7 DNA polymerase on duplex DNA

Publisher: Proceedings of the National Academy of Sciences

Date: 11-01-2010

DOI: 10.1073/PNAS.0912664107

Abstract: The DNA polymerases involved in DNA replication achieve high processivity of nucleotide incorporation by forming a complex with processivity factors. A model system for replicative DNA polymerases, the bacteriophage T7 DNA polymerase (gp5), encoded by gene 5, forms a tight, 1∶1 complex with Escherichia coli thioredoxin. By a mechanism that is not fully understood, thioredoxin acts as a processivity factor and converts gp5 from a distributive polymerase into a highly processive one. We use a single-molecule imaging approach to visualize the interaction of fluorescently labeled T7 DNA polymerase with double-stranded DNA. We have observed T7 gp5, both with and without thioredoxin, binding nonspecifically to double-stranded DNA and diffusing along the duplex. The gp5/thioredoxin complex remains tightly bound to the DNA while diffusing, whereas gp5 without thioredoxin undergoes frequent dissociation from and rebinding to the DNA. These observations suggest that thioredoxin increases the processivity of T7 DNA polymerase by suppressing microscopic hopping on and off the DNA and keeping the complex tightly bound to the duplex.

Highly‐Parallel Microfluidics‐Based Force Spectroscopy on Single Cytoskeletal Motors

Publisher: Wiley

Date: 23-03-2021

DOI: 10.1002/SMLL.202007388

An optical study of single pentacene molecules in n-tetradecane

Publisher: Elsevier BV

Date: 02-2000

DOI: 10.1016/S0009-2614(99)01393-7

Simple model for the power-law blinking of single semiconductor nanocrystals

Publisher: American Physical Society (APS)

Date: 06-12-2002

DOI: 10.1103/PHYSREVB.66.233202

Hopping of a processivity factor on DNA revealed by single-molecule assays of diffusion

Publisher: Proceedings of the National Academy of Sciences

Date: 05-08-2008

DOI: 10.1073/PNAS.0802676105

Abstract: Many DNA-interacting proteins diffuse on DNA to perform their biochemical functions. Processivity factors diffuse on DNA to permit unimpeded elongation by their associated DNA polymerases, but little is known regarding their rates and mechanisms of diffusion. The processivity factor of herpes simplex virus DNA polymerase, UL42, unlike “sliding cl ” processivity factors that normally form rings around DNA, binds DNA directly and tightly as a monomer, but can still diffuse on DNA. To investigate the mechanism of UL42 diffusion on DNA, we examined the effects of salt concentration on diffusion coefficient. Ensemble studies, employing electrophoretic mobility shift assays on relatively short DNAs, showed that off-rates of UL42 from DNA depended on DNA length at higher but not lower salt concentrations, consistent with the diffusion coefficient being salt-dependent. Direct assays of the motion of single fluorescently labeled UL42 molecules along DNA revealed increased diffusion at higher salt concentrations. Remarkably, the diffusion coefficients observed in these assays were ≈10 4 -fold higher than those calculated from ensemble experiments. Discrepancies between the single-molecule and ensemble results were resolved by the observation, in single-molecule experiments, that UL42 releases relatively slowly from the ends of DNA in a salt-dependent manner. The results indicate that UL42 “hops” rather than “slides,” i.e., it microscopically dissociates from and reassociates with DNA as it diffuses rather than remaining so intimately associated with DNA that cation condensation on the phosphate backbone does not affect its motion. These findings may be relevant to mechanisms of other processivity factors and DNA-binding proteins.

The transcription-repair coupling factor Mfd associates with RNA polymerase in the absence of exogenous damage

Publisher: Springer Science and Business Media LLC

Date: 20-04-2018

DOI: 10.1038/S41467-018-03790-Z

Abstract: During transcription elongation, bacterial RNA polymerase (RNAP) can pause, backtrack or stall when transcribing template DNA. Stalled transcription elongation complexes at sites of bulky lesions can be rescued by the transcription terminator Mfd. The molecular mechanisms of Mfd recruitment to transcription complexes in vivo remain to be elucidated, however. Using single-molecule live-cell imaging, we show that Mfd associates with elongation transcription complexes even in the absence of exogenous genotoxic stresses. This interaction requires an intact RNA polymerase-interacting domain of Mfd. In the presence of drugs that stall RNAP, we find that Mfd associates pervasively with RNAP. The residence time of Mfd foci reduces from 30 to 18 s in the presence of endogenous UvrA, suggesting that UvrA promotes the resolution of Mfd-RNAP complexes on DNA. Our results reveal that RNAP is frequently rescued by Mfd during normal growth and highlight a ubiquitous house-keeping role for Mfd in regulating transcription elongation.

Single-molecule live-cell imaging visualizes parallel pathways of prokaryotic nucleotide excision repair

Publisher: Springer Science and Business Media LLC

Date: 20-03-2020

DOI: 10.1038/S41467-020-15179-Y

Abstract: In the model organism Escherichia coli , helix distorting lesions are recognized by the UvrAB damage surveillance complex in the global genomic nucleotide excision repair pathway (GGR). Alternately, during transcription-coupled repair (TCR), UvrA is recruited to Mfd at sites of RNA polymerases stalled by lesions. Ultimately, damage recognition is mediated by UvrA, followed by verification by UvrB. Here we characterize the differences in the kinetics of interactions of UvrA with Mfd and UvrB by following functional, fluorescently tagged UvrA molecules in live TCR-deficient or wild-type cells. The lifetimes of UvrA in Mfd-dependent or Mfd-independent interactions in the absence of exogenous DNA damage are comparable in live cells, and are governed by UvrB. Upon UV irradiation, the lifetimes of UvrA strongly depended on, and matched those of Mfd. Overall, we illustrate a non-perturbative, imaging-based approach to quantify the kinetic signatures of damage recognition enzymes participating in multiple pathways in cells.

Author response: Single-molecule visualization of fast polymerase turnover in the bacterial replisome

Publisher: eLife Sciences Publications, Ltd

Date: 15-04-2017

DOI: 10.7554/ELIFE.23932.027

Continuous-wave two-photon excitation of individual CdS nanocrystallites

Publisher: AIP Publishing

Date: 06-08-2001

DOI: 10.1063/1.1391231

Abstract: By use of low-temperature confocal microscopy, continuous-wave two-photon fluorescence images are obtained of in idual CdS nanocrystallites embedded in a polymer matrix. The quadratic dependence of the emission rate on the applied laser power proves that the observed fluorescence originates from the simultaneous absorption of two photons. From the experimental data the two-photon absorption cross-section σ(2) could be determined, resulting in a value smaller than that known from literature. The work presented is a first step towards high-resolution fluorescence-excitation spectroscopy on the electronic states in the band edge, inaccessible by conventional one-photon spectroscopy.

Diatrack particle tracking software: Review of applications and performance evaluation

Publisher: Wiley

Date: 23-10-2017

DOI: 10.1111/TRA.12530

Specialised DNA polymerases in Escherichia coli: roles within multiple pathways

Publisher: Springer Science and Business Media LLC

Date: 26-04-2018

DOI: 10.1007/S00294-018-0840-X

Abstract: In many bacterial species, DNA damage triggers the SOS response a pathway that regulates the production of DNA repair and damage tolerance proteins, including error-prone DNA polymerases. These specialised polymerases are capable of bypassing lesions in the template DNA, a process known as translesion synthesis (TLS). Specificity for lesion types varies considerably between the different types of TLS polymerases. TLS polymerases are mainly described as working in the context of replisomes that are stalled at lesions or in lesion-containing gaps left behind the replisome. Recently, a series of single-molecule fluorescence microscopy studies have revealed that two TLS polymerases, pol IV and pol V, rarely colocalise with replisomes in Escherichia coli cells, suggesting that most TLS activity happens in a non-replisomal context. In this review, we re-visit the evidence for the involvement of TLS polymerases in other pathways. A series of genetic and biochemical studies indicates that TLS polymerases could participate in nucleotide excision repair, homologous recombination and transcription. In addition, oxidation of the nucleotide pool, which is known to be induced by multiple stressors, including many antibiotics, appears to favour TLS polymerase activity and thus increases mutation rates. Ultimately, participation of TLS polymerases within non-replisomal pathways may represent a major source of mutations in bacterial cells and calls for more extensive investigation.

Nudging Through a Nucleosome

Publisher: American Association for the Advancement of Science (AAAS)

Date: 31-07-2009

DOI: 10.1126/SCIENCE.1177311

Abstract: Single-molecule data suggest that RNA polymerase II moves a small step forward only when its DNA template briefly unwraps from the histone core.

Selective Bypass of a Lagging Strand Roadblock by the Eukaryotic Replicative DNA Helicase

Publisher: Elsevier BV

Date: 09-2011

DOI: 10.1016/J.CELL.2011.07.045

Spectroscopy on the B850 Band of Individual Light-Harvesting 2 Complexes of Rhodopseudomonas acidophila I. Experiments and Monte Carlo Simulations

Publisher: Elsevier BV

Date: 03-2001

DOI: 10.1016/S0006-3495(01)76132-2

Single-molecule visualization of Saccharomyces cerevisiae leading-strand synthesis reveals dynamic interaction between MTC and the replisome

Publisher: Proceedings of the National Academy of Sciences

Date: 18-09-2017

DOI: 10.1073/PNAS.1711291114

Abstract: Replication of genomic DNA is essential to all cells. The replisome, the multiprotein machine that performs DNA replication, contains many moving parts, the actions of which are poorly understood. Unraveling the dynamic behavior of these proteins requires novel application of single-molecule imaging techniques to eliminate averaging inherent in ensemble methods and to directly observe short-lived events. Here, we present single-molecule observations of an active Saccharomyces cerevisiae replisome using purified proteins. We find that a checkpoint complex (Mrc1–Tof1–Csm3), known to bind and to speed up the replisome, interacts only transiently with the replisome. This work represents a major step toward establishing the tools needed to understand the detailed kinetics of proteins within the complex eukaryotic replisome.

Membrane-on-a-Chip: Microstructured Silicon/Silicon-Dioxide Chips for High-Throughput Screening of Membrane Transport and Viral Membrane Fusion

Publisher: American Chemical Society (ACS)

Date: 11-03-2014

DOI: 10.1021/NN405884A

Abstract: Screening of transport processes across biological membranes is hindered by the challenge to establish fragile supported lipid bilayers and the difficulty to determine at which side of the membrane reactants reside. Here, we present a method for the generation of suspended lipid bilayers with physiological relevant lipid compositions on microstructured Si/SiO2 chips that allow for high-throughput screening of both membrane transport and viral membrane fusion. Simultaneous observation of hundreds of single-membrane channels yields statistical information revealing population heterogeneities of the pore assembly and conductance of the bacterial toxin α-hemolysin (αHL). The influence of lipid composition and ionic strength on αHL pore formation was investigated at the single-channel level, resolving features of the pore-assembly pathway. Pore formation is inhibited by a specific antibody, demonstrating the applicability of the platform for drug screening of bacterial toxins and cell-penetrating agents. Furthermore, fusion of H3N2 influenza viruses with suspended lipid bilayers can be observed directly using a specialized chip architecture. The presented micropore arrays are compatible with fluorescence readout from below using an air objective, thus allowing high-throughput screening of membrane transport in multiwell formats in analogy to plate readers.

Analysis of Kinetic Intermediates in Single-Particle Dwell-Time Distributions

Publisher: Elsevier BV

Date: 07-2010

DOI: 10.1016/J.BPJ.2010.04.049

Cooperative Chikungunya virus membrane fusion and its sub-stoichiometric inhibition by CHK-152 antibody

Publisher: Cold Spring Harbor Laboratory

Date: 19-03-2020

DOI: 10.1101/2020.03.18.996512

Abstract: Chikungunya virus (CHIKV) presents a major burden on healthcare systems worldwide, but specific treatment remains unavailable. Attachment and fusion of CHIKV to the host cell membrane is mediated by the E1/E2 protein spikes. We used an in vitro single-particle fusion assay to study the effect of the potent, neutralizing antibody CHK-152 on CHIKV binding and fusion. We find that CHK-152 shields the virions, inhibiting interaction with the target membrane and inhibiting fusion. Analysis of the ratio of bound antibodies to epitopes implied that CHIKV fusion is a highly cooperative process. Further, dissociation of the antibody at lower pH results in a finely balanced kinetic competition between inhibition and fusion, suggesting a window of opportunity for the spike proteins to act and mediate fusion even in the presence of antibody.

Single-molecule kinetics of lambda exonuclease reveal base dependence and dynamic disorder

Publisher: American Association for the Advancement of Science (AAAS)

Date: 29-08-2003

DOI: 10.1126/SCIENCE.1084387

Abstract: We used a multiplexed approach based on flow-stretched DNA to monitor the enzymatic digestion of λ-phage DNA by in idual bacteriophage λ exonuclease molecules. Statistical analyses of multiple single-molecule trajectories observed simultaneously reveal that the catalytic rate is dependent on the local base content of the substrate DNA. By relating single-molecule kinetics to the free energies of hydrogen bonding and base stacking, we establish that the melting of a base from the DNA is the rate-limiting step in the catalytic cycle. The catalytic rate also exhibits large fluctuations independent of the sequence, which we attribute to conformational changes of the enzyme-DNA complex.

Chikungunya virus fusion properties elucidated by single-particle and bulk approaches

Publisher: Microbiology Society

Date: 08-2015

DOI: 10.1099/VIR.0.000144

Abstract: Chikungunya virus (CHIKV) is a rapidly spreading, enveloped alphavirus causing fever, rash and debilitating polyarthritis. No specific treatment or vaccines are available to treat or prevent infection. For the rational design of vaccines and antiviral drugs, it is imperative to understand the molecular mechanisms involved in CHIKV infection. A critical step in the life cycle of CHIKV is fusion of the viral membrane with a host cell membrane. Here, we elucidate this process using ensemble-averaging liposome-virus fusion studies, in which the fusion behaviour of a large virus population is measured, and a newly developed microscopy-based single-particle assay, in which the fusion kinetics of an in idual particle can be visualised. The combination of these approaches allowed us to obtain detailed insight into the kinetics, lipid dependency and pH dependency of hemifusion. We found that CHIKV fusion is strictly dependent on low pH, with a threshold of pH 6.2 and optimal fusion efficiency below pH 5.6. At this pH, CHIKV fuses rapidly with target membranes, with typically half of the fusion occurring within 2 s after acidification. Cholesterol and sphingomyelin in the target membrane were found to strongly enhance the fusion process. By analysing our single-particle data using kinetic models, we were able to deduce that the number of rate-limiting steps occurring before hemifusion equals about three. To explain these data, we propose a mechanistic model in which multiple E1 fusion trimers are involved in initiating the fusion process.

Visualizing DNA Replication at the Single-Molecule Level

Publisher: Elsevier

Date: 2010

DOI: 10.1016/S0076-6879(10)75011-4

Influenza-virus membrane fusion by cooperative fold-back of stochastically induced hemagglutinin intermediates

Publisher: eLife Sciences Publications, Ltd

Date: 19-02-2013

DOI: 10.7554/ELIFE.00333

Abstract: Influenza virus penetrates cells by fusion of viral and endosomal membranes catalyzed by the viral hemagglutinin (HA). Structures of the initial and final states of the HA trimer define the fusion endpoints, but do not specify intermediates. We have characterized these transitions by analyzing low-pH-induced fusion kinetics of in idual virions and validated the analysis by computer simulation. We detect initial engagement with the target membrane of fusion peptides from independently triggered HAs within the larger virus-target contact patch fusion then requires engagement of three or four neighboring HA trimers. Effects of mutations in HA indicate that withdrawal of the fusion peptide from a pocket in the pre-fusion trimer is rate-limiting for both events, but the requirement for cooperative action of several HAs to bring the fusing membranes together leads to a long-lived intermediate state for single, extended HA trimers. This intermediate is thus a fundamental aspect of the fusion mechanism.

External control of reactions in microdroplets

Publisher: Springer Science and Business Media LLC

Date: 02-07-2015

DOI: 10.1038/SREP11837

Abstract: Scale reduction of chemical reactions enables novel screening and synthesis approaches that facilitate a highly parallelized and combinatorial exploration of chemical space. Droplet-based microfluidics have evolved as a powerful platform to allow many chemical reactions within small volumes that each can be controlled and manipulated. A significant technical challenge is the ability to change the concentration of reactants inside a droplet. Here we describe a strategy that relies on the use of reactants that are soluble in both oil and water and allow a passive, diffusive exchange of reactants between the oil and aqueous phases to externally control composition of the droplets. We demonstrate the applicability of our approach by externally changing the pH inside microdroplets without the need for physical manipulation or droplet merging.

Author response: Spatial and temporal organization of RecA in the Escherichia coli DNA-damage response

Publisher: eLife Sciences Publications, Ltd

Date: 08-01-2019

DOI: 10.7554/ELIFE.42761.026

Detection of kinetic change points in piece-wise linear single molecule motion

Publisher: AIP Publishing

Date: 03-01-2018

DOI: 10.1063/1.5009387

Abstract: Single-molecule approaches present a powerful way to obtain detailed kinetic information at the molecular level. However, the identification of small rate changes is often hindered by the considerable noise present in such single-molecule kinetic data. We present a general method to detect such kinetic change points in trajectories of motion of processive single molecules having Gaussian noise, with a minimum number of parameters and without the need of an assumed kinetic model beyond piece-wise linearity of motion. Kinetic change points are detected using a likelihood ratio test in which the probability of no change is compared to the probability of a change occurring, given the experimental noise. A predetermined confidence interval minimizes the occurrence of false detections. Applying the method recursively to all sub-regions of a single molecule trajectory ensures that all kinetic change points are located. The algorithm presented allows rigorous and quantitative determination of kinetic change points in noisy single molecule observations without the need for filtering or binning, which reduce temporal resolution and obscure dynamics. The statistical framework for the approach and implementation details are discussed. The detection power of the algorithm is assessed using simulations with both single kinetic changes and multiple kinetic changes that typically arise in observations of single-molecule DNA-replication reactions. Implementations of the algorithm are provided in ImageJ plugin format written in Java and in the Julia language for numeric computing, with accompanying Jupyter Notebooks to allow reproduction of the analysis presented here.

Single-Molecule Specific Mislocalization of Red Fluorescent Proteins in Live Escherichia coli

Publisher: Elsevier BV

Date: 07-2016

DOI: 10.1016/J.BPJ.2016.05.047

Identification of Multiple Kinetic Populations of DNA-Binding Proteins in Live Cells

Publisher: Elsevier BV

Date: 09-2019

DOI: 10.1016/J.BPJ.2019.07.015

A single-molecule view of DNA replication: the dynamic nature of multi-protein complexes revealed

Publisher: Elsevier BV

Date: 10-2013

DOI: 10.1016/J.SBI.2013.06.018

Abstract: Recent advances in the development of single-molecule approaches have made it possible to study the dynamics of biomolecular systems in great detail. More recently, such tools have been applied to study the dynamic nature of large multi-protein complexes that support multiple enzymatic activities. In this review, we will discuss single-molecule studies of the replisome, the protein complex responsible for the coordinated replication of double-stranded DNA. In particular, we will focus on new insights obtained into the dynamic nature of the composition of the DNA-replication machinery and how the dynamic replacement of components plays a role in the regulation of the DNA-replication process.

Plasticity of Multi-Protein Complexes

Publisher: Elsevier BV

Date: 10-2018

DOI: 10.1016/J.JMB.2018.08.008

Speeding up biomolecular interactions by molecular sledding

Publisher: Royal Society of Chemistry (RSC)

Date: 2016

DOI: 10.1039/C5SC03063C

Abstract: Reaction partners are functionalised with a DNA sliding peptide and the association between them is significantly speeded up in the presence of DNA in solution.

Single-molecule live-cell imaging of clathrin-based endocytosis.

Publisher: Portland Press Ltd.

Date: 2005

DOI: 10.1042/BSS0720071

Abstract: Clathrin-coated vesicles carry traffic from the plasma membrane to endosomes. We report here the first real-time visualization of cargo sorting and endocytosis by clathrin-coated pits in living cells. We have visualized the formation of coats by monitoring the incorporation of fluorescently tagged clathrin or its adaptor AP-2 (adaptor protein 2), and have followed clathrin-mediated uptake of transferrin, single LDL (low-density lipoprotein) and single reovirus particles. The intensity of a cargo-loaded clathrin cluster grows steadily during its lifetime, and the time required to complete assembly is proportional to the size of the cargo particle. These results are consistent with a nucleation-growth mechanism and an approximately constant growth rate. There are no preferred nucleation sites. A proportion of the nucleation events appear to be abortive. Cargo incorporation occurs primarily or exclusively in a newly formed coated pit, and loading appears to commit that pit to finish assembly. Our data led to a model in which coated pits initiate randomly, but collapse with high likelihood unless stabilized, presumably by cargo capture.

Far-field fluorescence microscopy beyond the diffraction limit

Publisher: Optica Publishing Group

Date: 04-1999

DOI: 10.1364/JOSAA.16.000909

Spectroscopy of individual LH2 complexes of Rhodopseudomonas acidophila: localized excitations in the B800 band

Publisher: Elsevier BV

Date: 08-1999

DOI: 10.1016/S0301-0104(99)00105-6

Single-molecule studies of fork dynamics in Escherichia coli DNA replication

Publisher: Springer Science and Business Media LLC

Date: 27-01-2008

DOI: 10.1038/NSMB.1381

Residues in the central β-hairpin of the DNA helicase of bacteriophage T7 are important in DNA unwinding

Publisher: Proceedings of the National Academy of Sciences

Date: 29-03-2010

DOI: 10.1073/PNAS.1002734107

Abstract: The ring-shaped helicase of bacteriophage T7 (gp4), the product of gene 4, has basic β-hairpin loops lining its central core where they are postulated to be the major sites of DNA interaction. We have altered multiple residues within the β-hairpin loop to determine their role during dTTPase-driven DNA unwinding. Residues His-465, Leu-466, and Asn-468 are essential for both DNA unwinding and DNA synthesis mediated by T7 DNA polymerase during leading-strand DNA synthesis. Gp4-K467A, gp4-K471A, and gp4-K473A form fewer hexamers than heptamers compared to wild-type helicase and alone are deficient in DNA unwinding. However, they complement for the growth of T7 bacteriophage lacking gene 4. Single-molecule studies show that these three altered helicases support rates of leading-strand DNA synthesis comparable to that observed with wild-type gp4. Gp4-K467A, devoid of unwinding activity alone, supports leading-strand synthesis in the presence of T7 DNA polymerase. We propose that DNA polymerase limits the backward movement of the helicase during unwinding as well as assisting the forward movement necessary for strand separation.

Kinetics of proton transport into influenza virions by the viral M2 channel.

Publisher: Public Library of Science (PLoS)

Date: 06-03-2012

DOI: 10.1371/JOURNAL.PONE.0031566

Real-time single-molecule observation of chaperone-assisted protein folding

Publisher: American Association for the Advancement of Science (AAAS)

Date: 14-12-2022

DOI: 10.1126/SCIADV.ADD0922

Abstract: The ability of heat shock protein 70 (Hsp70) molecular chaperones to remodel the conformation of their clients is central to their biological function however, questions remain regarding the precise molecular mechanisms by which Hsp70 machinery interacts with the client and how this contributes toward efficient protein folding. Here, we used total internal reflection fluorescence (TIRF) microscopy and single-molecule fluorescence resonance energy transfer (smFRET) to temporally observe the conformational changes that occur to in idual firefly luciferase proteins as they are folded by the bacterial Hsp70 system. We observed multiple cycles of chaperone binding and release to an in idual client during refolding and determined that high rates of chaperone cycling improves refolding yield. Furthermore, we demonstrate that DnaJ remodels misfolded proteins via a conformational selection mechanism, whereas DnaK resolves misfolded states via mechanical unfolding. This study illustrates that the temporal observation of chaperone-assisted folding enables the elucidation of key mechanistic details inaccessible using other approaches.

Bacterial replication, transcription and translation: mechanistic insights from single-molecule biochemical studies

Publisher: Springer Science and Business Media LLC

Date: 03-04-2013

DOI: 10.1038/NRMICRO2994

Abstract: Decades of research have resulted in a remarkably detailed understanding of the molecular mechanisms of bacterial DNA replication, transcription and translation. Our understanding of the kinetics and physical mechanisms that drive these processes forward has been expanded by the ability of single-molecule in vitro techniques, such as force spectroscopy and single-molecule Förster (fluorescence) resonance energy transfer (smFRET), to capture short-lived intermediate states in complex pathways. Furthermore, these technologies have revealed novel mechanisms that support enzyme processivity and govern the assembly of large multicomponent complexes. Here, we summarize the application of in vitro single-molecule studies to the analysis of fundamental bacterial processes, with a focus on the most recent functional insights that have been gained from fluorescence-based methods.

Uncoupling of Sister Replisomes during Eukaryotic DNA Replication

Publisher: Elsevier BV

Date: 12-2010

DOI: 10.1016/J.MOLCEL.2010.11.027

Single-molecule live-cell imaging of bacterial DNA repair and damage tolerance

Publisher: Portland Press Ltd.

Date: 12-2017

DOI: 10.1042/BST20170055

Abstract: Genomic DNA is constantly under threat from intracellular and environmental factors that damage its chemical structure. Uncorrected DNA damage may impede cellular propagation or even result in cell death, making it critical to restore genomic integrity. Decades of research have revealed a wide range of mechanisms through which repair factors recognize damage and co-ordinate repair processes. In recent years, single-molecule live-cell imaging methods have further enriched our understanding of how repair factors operate in the crowded intracellular environment. The ability to follow in idual biochemical events, as they occur in live cells, makes single-molecule techniques tremendously powerful to uncover the spatial organization and temporal regulation of repair factors during DNA–repair reactions. In this review, we will cover practical aspects of single-molecule live-cell imaging and highlight recent advances accomplished by the application of these experimental approaches to the study of DNA–repair processes in prokaryotes.

Spectroscopy of Individual Light-Harvesting 2 Complexes of Rhodopseudomonas acidophila: Diagonal Disorder, Intercomplex Heterogeneity, Spectral Diffusion, and Energy Transfer in the B800 Band

Publisher: Elsevier BV

Date: 03-2000

DOI: 10.1016/S0006-3495(00)76709-9

Single-molecule studies of the replisome

Publisher: Annual Reviews

Date: 04-2010

DOI: 10.1146/ANNUREV.BIOPHYS.093008.131327

Abstract: Replication of DNA is carried out by the replisome, a multiprotein complex responsible for the unwinding of parental DNA and the synthesis of DNA on each of the two DNA strands. The impressive speed and processivity with which the replisome duplicates DNA are a result of a set of tightly regulated interactions between the replication proteins. The transient nature of these protein interactions makes it challenging to study the dynamics of the replisome by ensemble-averaging techniques. This review describes single-molecule methods that allow the study of in idual replication proteins and their functioning within the replisome. The ability to mechanically manipulate in idual DNA molecules and record the dynamic behavior of the replisome while it duplicates DNA has led to an improved understanding of the molecular mechanisms underlying DNA replication.

Anisotropic functionalization of upconversion nanoparticles

Publisher: Royal Society of Chemistry (RSC)

Date: 2018

DOI: 10.1039/C8SC01023D

Abstract: Ligand competition directs heterogeneous bio-chemistry surface and self-assembly for upconversion nanoparticles.

Droplet microfluidics: A tool for biology, chemistry and nanotechnology

Publisher: Elsevier BV

Date: 09-2016

DOI: 10.1016/J.TRAC.2016.05.019

Mechanisms of influenza viral membrane fusion

Publisher: Elsevier BV

Date: 12-2016

DOI: 10.1016/J.SEMCDB.2016.07.007

Abstract: Influenza viral particles are enveloped by a lipid bilayer. A major step in infection is fusion of the viral and host cellular membranes, a process with large kinetic barriers. Influenza membrane fusion is catalyzed by hemagglutinin (HA), a class I viral fusion protein activated by low pH. The exact nature of the HA conformational changes that deliver the energy required for fusion remains poorly understood. This review summarizes our current knowledge of HA structure and dynamics, describes recent single-particle experiments and modeling studies, and discusses their role in understanding how multiple HAs mediate fusion. These approaches provide a mechanistic picture in which HAs independently and stochastically insert into the target membrane, forming a cluster of HAs that is collectively able to overcome the barrier to membrane fusion. The new experimental and modeling approaches described in this review hold promise for a more complete understanding of other viral fusion systems and the protein systems responsible for cellular fusion.

A direct proofreader-clamp interaction stabilizes the Pol III replicase in the polymerization mode

Publisher: Wiley

Date: 22-02-2013

DOI: 10.1038/EMBOJ.2012.347

Global maps of soil temperature.

Publisher: Wiley

Date: 11-02-2022

DOI: 10.1111/GCB.16060

Abstract: Research in global change ecology relies heavily on global climatic grids derived from estimates of air temperature in open areas at around 2 m above the ground. These climatic grids do not reflect conditions below vegetation canopies and near the ground surface, where critical ecosystem functions occur and most terrestrial species reside. Here, we provide global maps of soil temperature and bioclimatic variables at a 1-km

Author response: A mechanism for the extension and unfolding of parallel telomeric G-quadruplexes by human telomerase at single-molecule resolution

Publisher: eLife Sciences Publications, Ltd

Date: 28-07-2020

DOI: 10.7554/ELIFE.56428.SA2

Recycling of single-stranded DNA-binding protein by the bacterial replisome

Publisher: Oxford University Press (OUP)

Date: 15-02-2019

DOI: 10.1093/NAR/GKZ090

Unsynchronised subunit motion in single trimeric sodium-coupled aspartate transporters

Publisher: Springer Science and Business Media LLC

Date: 10-2013

DOI: 10.1038/NATURE12538

Abstract: Excitatory amino acid transporters (EAATs) are secondary transport proteins that mediate the uptake of glutamate and other amino acids. EAATs fulfil an important role in neuronal signal transmission by clearing the excitatory neurotransmitters from the synaptic cleft after depolarization of the postsynaptic neuron. An intensively studied model system for understanding the transport mechanism of EAATs is the archaeal aspartate transporter GltPh. Each subunit in the homotrimeric GltPh supports the coupled translocation of one aspartate molecule and three Na(+) ions as well as an uncoupled flux of Cl(-) ions. Recent crystal structures of GltPh revealed three possible conformations for the subunits, but it is unclear whether the motions of in idual subunits are coordinated to support transport. Here, we report the direct observation of conformational dynamics in in idual GltPh trimers embedded in the membrane by applying single-molecule fluorescence resonance energy transfer (FRET). By analysing the transporters in a lipid bilayer instead of commonly used detergent micelles, we achieve conditions that approximate the physiologically relevant ones. From the kinetics of FRET level transitions we conclude that the three GltPh subunits undergo conformational changes stochastically and independently of each other.

Real-time dynamic single-molecule protein sequencing on an integrated semiconductor device.

Publisher: American Association for the Advancement of Science (AAAS)

Date: 14-10-2022

DOI: 10.1126/SCIENCE.ABO7651

Abstract: Studies of the proteome would benefit greatly from methods to directly sequence and digitally quantify proteins and detect posttranslational modifications with single-molecule sensitivity. Here, we demonstrate single-molecule protein sequencing using a dynamic approach in which single peptides are probed in real time by a mixture of dye-labeled N-terminal amino acid recognizers and simultaneously cleaved by aminopeptidases. We annotate amino acids and identify the peptide sequence by measuring fluorescence intensity, lifetime, and binding kinetics on an integrated semiconductor chip. Our results demonstrate the kinetic principles that allow recognizers to identify multiple amino acids in an information-rich manner that enables discrimination of single amino acid substitutions and posttranslational modifications. With further development, we anticipate that this approach will offer a sensitive, scalable, and accessible platform for single-molecule proteomic studies and applications.

Single-molecule studies of fork dynamics in Escherichia coli DNA replication (Nature Structural and Molecular Biology (2008) 15, (170-176))

Publisher: Springer Science and Business Media LLC

Date: 08-2107

DOI: 10.1038/NSMB0908-998A

RecFOR epistasis group: RecF and RecO have distinct localizations and functions in Escherichia coli

Publisher: Oxford University Press (OUP)

Date: 18-01-2019

DOI: 10.1093/NAR/GKZ003

Method for measurement of viral fusion kinetics at the single particle level

Publisher: MyJove Corporation

Date: 07-09-2009

DOI: 10.3791/1484

Nuclease dead Cas9 is a programmable roadblock for DNA replication

Publisher: Springer Science and Business Media LLC

Date: 16-09-2019

DOI: 10.1038/S41598-019-49837-Z

Abstract: Limited experimental tools are available to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct as a generic, novel, targetable protein–DNA roadblock for studying mechanisms underlying enzymatic activities on DNA substrates in vitro . We illustrate the broad utility of this tool by demonstrating replication fork arrest by the specifically bound dCas9–guideRNA complex to arrest viral, bacterial and eukaryotic replication forks in vitro .

Single-molecule live-cell imaging visualizes parallel pathways of prokaryotic nucleotide excision repair

Publisher: Cold Spring Harbor Laboratory

Date: 08-01-2019

DOI: 10.1101/515502

Abstract: In the model organism Escherichia coli , helix distorting lesions are recognized by the UvrAB damage surveillance complex in the global genomic nucleotide excision repair pathway (GGR). Alternately, during transcription-coupled repair (TCR), UvrA is recruited to Mfd at sites of RNA polymerases stalled or paused by lesions. Ultimately, damage recognition is mediated by UvrA, culminating in the loading of the damage verification enzyme UvrB. We set out to characterize the differences in the kinetics of interactions of UvrA with Mfd and UvrB. We followed functional, fluorescently tagged UvrA molecules in live cells and measured their residence times in TCR-deficient or wild-type cells. We demonstrate that the lifetimes of UvrA in Mfd-dependent or Mfd-independent interactions in the absence of exogenous DNA damage are comparable in live cells, and are governed by UvrB. Upon UV irradiation, we found that the lifetimes of UvrA strongly depended on, and matched those of Mfd. Here, we illustrate a non-perturbative, imaging-based approach to quantify the kinetic signatures of damage recognition enzymes participating in multiple pathways in cells.

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

Publisher: MyJove Corporation

Date: 09-10-2009

DOI: 10.3791/1529

A Tool for Alignment and Averaging of Sparse Fluorescence Signals in Rod-Shaped Bacteria

Publisher: Elsevier BV

Date: 04-2016

DOI: 10.1016/J.BPJ.2016.02.039

mKikGR, a Monomeric Photoswitchable Fluorescent Protein

Publisher: Public Library of Science (PLoS)

Date: 15-12-2008

DOI: 10.1371/JOURNAL.PONE.0003944

An α-cyanostilbene derivative for the enhanced detection and imaging of amyloid fibril aggregates

Publisher: Cold Spring Harbor Laboratory

Date: 27-06-2020

DOI: 10.1101/2020.06.25.172627

Abstract: The aggregation of proteins into amyloid fibrils has been implicated in the pathogenesis of a variety of neurodegenerative diseases, including Alzheimer’s and Parkinson’s disease. Benzothiazole dyes such as Thioflavin T (ThT) are well characterised and widely used fluorescent probes for monitoring amyloid fibril formation. However, existing dyes lack sensitivity and specificity to oligomeric intermediates formed during fibril formation. In this work we describe the use of an α-cyanostilbene derivative with aggregation-induced emission properties (called ASCP) as a fluorescent probe for the detection of amyloid fibrils. Similar to ThT, ASCP is fluorogenic in the presence of amyloid fibrils and upon binding and excitation at 460 nm produces a red-shifted emission with a large Stokes shift of 145 nm. ASCP has a higher binding affinity to fibrillar α-synuclein than ThT and likely shares the same binding sites to amyloid fibrils. Importantly, ASCP was found to also be fluorogenic in the presence of amorphous aggregates and can detect oligomeric species formed early during aggregation. Moreover, ASCP can be used to visualise fibrils via Total Internal Reflection Fluorescence (TIRF) microscopy and, due to its large Stokes shift, simultaneously monitor the fluorescence emission of other labelled proteins following excitation with the same laser used to excite ASCP. Consequently, ASCP possesses enhanced and unique spectral characteristics compared to ThT that make it a promising alternative for the in vitro study of amyloid fibrils and the mechanisms by which they form.

Single-Molecule Observation of Prokaryotic DNA Replication

Publisher: Humana Press

Date: 2009

DOI: 10.1007/978-1-60327-815-7_22

A single-molecule characterization of p53 search on DNA

Publisher: Proceedings of the National Academy of Sciences

Date: 22-12-2011

DOI: 10.1073/PNAS.1016020107

Abstract: The tumor suppressor p53 slides along DNA while searching for its cognate site. Central to this process is the basic C-terminal domain, whose regulatory role and its coordination with the core DNA-binding domain is highly debated. Here we use single-molecule techniques to characterize the search process and disentangle the roles played by these two DNA-binding domains in the search process. We demonstrate that the C-terminal domain is capable of rapid translocation, while the core domain is unable to slide and instead hops along DNA. These findings are integrated into a model, in which the C-terminal domain mediates fast sliding of p53, while the core domain s les DNA by frequent dissociation and reassociation, allowing for rapid scanning of long DNA regions. The model further proposes how modifications of the C-terminal domain can activate “latent” p53 and reconciles seemingly contradictory data on the action of different domains and their coordination.

The E. coli helicase does not use ATP during replication

Publisher: Cold Spring Harbor Laboratory

Date: 07-07-2021

DOI: 10.1101/2021.07.07.451541

Abstract: The replisome is responsible for replication of DNA in all domains of life, with several of its in idual enzyme components relying on hydrolysis of nucleoside triphosphates to provide energy for replisome function. Half a century of biochemical studies have demonstrated a dependence on ATP as an energy source for helicases to unwind duplex DNA during replication. Through single-molecule visualization of DNA replication by the Escherichia coli replisome, we demonstrate that the DnaB helicase does not rely on hydrolysis of ATP (or any ribo-NTPs) in the context of the elongating replisome. We establish that nucleotide incorporation by the leading-strand polymerase is the main motor driving the replication process. Polymerases provide the energy for helicase-mediated DNA unwinding during E. coli DNA replication.

The more the merrier: high-throughput single-molecule techniques

Publisher: Portland Press Ltd.

Date: 15-06-2017

DOI: 10.1042/BST20160137

Abstract: The single-molecule approach seeks to understand molecular mechanisms by observing biomolecular processes at the level of in idual molecules. These methods have led to a developing understanding that for many processes, a ersity of behaviours will be observed, representing a multitude of pathways. This realisation necessitates that an adequate number of observations are recorded to fully characterise this ersity. The requirement for large numbers of observations to adequately s le distributions, subpopulations, and rare events presents a significant challenge for single-molecule techniques, which by their nature do not typically provide very high throughput. This review will discuss many developing techniques which address this issue by combining nanolithographic approaches, such as zero-mode waveguides and DNA curtains, with single-molecule fluorescence microscopy, and by drastically increasing throughput of force-based approaches such as magnetic tweezers and laminar-flow techniques. These methods not only allow the collection of large volumes of single-molecule data in single experiments, but have also made improvements to ease-of-use, accessibility, and automation of data analysis.

Single-molecule visualization of fast polymerase turnover in the bacterial replisome

Publisher: eLife Sciences Publications, Ltd

Date: 22-04-2017

DOI: 10.7554/ELIFE.23932

Abstract: The Escherichia coli DNA replication machinery has been used as a road map to uncover design rules that enable DNA duplication with high efficiency and fidelity. Although the enzymatic activities of the replicative DNA Pol III are well understood, its dynamics within the replisome are not. Here, we test the accepted view that the Pol III holoenzyme remains stably associated within the replisome. We use in vitro single-molecule assays with fluorescently labeled polymerases to demonstrate that the Pol III* complex (holoenzyme lacking the β2 sliding cl ), is rapidly exchanged during processive DNA replication. Nevertheless, the replisome is highly resistant to dilution in the absence of Pol III* in solution. We further show similar exchange in live cells containing labeled cl loader and polymerase. These observations suggest a concentration-dependent exchange mechanism providing a balance between stability and plasticity, facilitating replacement of replisomal components dependent on their availability in the environment.

Theory of bimolecular reactions in a solution with linear traps: Application to the problem of target search on DNA

Publisher: American Physical Society (APS)

Date: 03-11-2015

DOI: 10.1103/PHYSREVE.92.052703

Single-Molecule Imaging of an in Vitro-Evolved RNA Aptamer Reveals Homogeneous Ligand Binding Kinetics

Publisher: American Chemical Society (ACS)

Date: 02-07-2009

DOI: 10.1021/JA901880V

Timing, coordination, and rhythm: Acrobatics at the DNA replication fork

Publisher: Elsevier BV

Date: 06-2010

DOI: 10.1074/JBC.R109.022939

Proliferating Cell Nuclear Antigen Uses Two Distinct Modes to Move along DNA

Publisher: Elsevier BV

Date: 06-2009

DOI: 10.1074/JBC.M109.008706

How DNA-repair proteins find their targets

Publisher: Proceedings of the National Academy of Sciences

Date: 11-10-2012

DOI: 10.1073/PNAS.1215845109

Using single-molecule approaches to understand the molecular mechanisms of heat-shock protein chaperone function

Publisher: Elsevier BV

Date: 10-2018

DOI: 10.1016/J.JMB.2018.05.021

Abstract: The heat-shock proteins (Hsp) are a family of molecular chaperones, which collectively form a network that is critical for the maintenance of protein homeostasis. Traditional ensemble-based measurements have provided a wealth of knowledge on the function of in idual Hsps and the Hsp network however, such techniques are limited in their ability to resolve the heterogeneous, dynamic and transient interactions that molecular chaperones make with their client proteins. Single-molecule techniques have emerged as a powerful tool to study dynamic biological systems, as they enable rare and transient populations to be identified that would usually be masked in ensemble measurements. Thus, single-molecule techniques are particularly amenable for the study of Hsps and have begun to be used to reveal novel mechanistic details of their function. In this review, we discuss the current understanding of the chaperone action of Hsps and how gaps in the field can be addressed using single-molecule methods. Specifically, this review focuses on the ATP-independent small Hsps and the broader Hsp network and describes how these dynamic systems are amenable to single-molecule techniques.

Single-molecule imaging reveals molecular coupling between transcription and DNA repair in live cells

Publisher: Cold Spring Harbor Laboratory

Date: 08-01-2019

DOI: 10.1101/515536

Abstract: Actively transcribed genes are preferentially repaired in a conserved repair reaction known as transcription-coupled nucleotide excision repair 1–3 . During this reaction, stalled transcription elongation complexes at sites of lesions serve as a signal to trigger the assembly of nucleotide excision repair factors (reviewed in ref. 4,5 ). In the model organism Escherichia coli , the transcription-repair coupling factor Mfd displaces the stalled RNA polymerase and hands-off the stall site to the nucleotide excision repair factors UvrAB for damage detection 6–9 . Despite in vitro evidence, it remains unclear how in live cells the stall site is faithfully handed over to UvrB from RNA polymerase and whether this handoff occurs via the Mfd-UvrA 2 -UvrB complex or via alternate reaction intermediates. Here, we visualise Mfd, the central player of transcription-coupled repair in actively growing cells and determine the catalytic requirements for faithful completion of the handoff during transcription-coupled repair. We find that the Mfd-UvrA 2 complex is arrested on DNA in the absence of UvrB. Further, Mfd-UvrA 2 -UvrB complexes formed by UvrB mutants deficient in DNA loading and damage recognition, were also impaired in successful handoff. Our observations demonstrate that in live cells, the dissociation of Mfd is tightly coupled to successful loading of UvrB, providing a mechanism via which loading of UvrB occurs in a strand-specific manner during transcription-coupled repair.

DNA replication at the single-molecule level

Publisher: Royal Society of Chemistry (RSC)

Date: 2014

DOI: 10.1039/C3CS60391A

Abstract: In this review, we highlight recent progress in single-molecule biophysics research that has led to exciting new findings about the mechanism of DNA replication.

Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited

Publisher: Springer Science and Business Media LLC

Date: 25-12-2005

DOI: 10.1038/NCHEMBIO759

Abstract: Enzymes are biological catalysts vital to life processes and have attracted century-long investigation. The classic Michaelis-Menten mechanism provides a highly satisfactory description of catalytic activities for large ensembles of enzyme molecules. Here we tested the Michaelis-Menten equation at the single-molecule level. We monitored long time traces of enzymatic turnovers for in idual beta-galactosidase molecules by detecting one fluorescent product at a time. A molecular memory phenomenon arises at high substrate concentrations, characterized by clusters of turnover events separated by periods of low activity. Such memory lasts for decades of timescales ranging from milliseconds to seconds owing to the presence of interconverting conformers with broadly distributed lifetimes. We proved that the Michaelis-Menten equation still holds even for a fluctuating single enzyme, but bears a different microscopic interpretation.

Watching cellular machinery in action, one molecule at a time

Publisher: Rockefeller University Press

Date: 15-12-2017

DOI: 10.1083/JCB.201610025

Abstract: Single-molecule manipulation and imaging techniques have become important elements of the biologist’s toolkit to gain mechanistic insights into cellular processes. By removing ensemble averaging, single-molecule methods provide unique access to the dynamic behavior of biomolecules. Recently, the use of these approaches has expanded to the study of complex multiprotein systems and has enabled detailed characterization of the behavior of in idual molecules inside living cells. In this review, we provide an overview of the various force- and fluorescence-based single-molecule methods with applications both in vitro and in vivo, highlighting these advances by describing their applications in studies on cytoskeletal motors and DNA replication. We also discuss how single-molecule approaches have increased our understanding of the dynamic behavior of complex multiprotein systems. These methods have shown that the behavior of multicomponent protein complexes is highly stochastic and less linear and deterministic than previously thought. Further development of single-molecule tools will help to elucidate the molecular dynamics of these complex systems both inside the cell and in solutions with purified components.

Honey, I shrunk the DNA: DNA length as a probe for nucleic‐acid enzyme activity

Publisher: Wiley

Date: 02-11-2006

DOI: 10.1002/BIP.20624

Abstract: The replication, recombination, and repair of DNA are processes essential for the maintenance of genomic information and require the activity of numerous enzymes that catalyze the polymerization or digestion of DNA. This review will discuss how differences in elastic properties between single- and double-stranded DNA can be used as a probe to study the dynamics of these enzymes at the single-molecule level.

Quantification of ligand density and stoichiometry on the surface of liposomes using single-molecule fluorescence imaging

Publisher: Elsevier BV

Date: 05-2018

DOI: 10.1016/J.JCONREL.2018.03.022

Abstract: Despite the longstanding existence of liposome technology in drug delivery applications, there have been no ligand-directed liposome formulations approved for clinical use to date. This lack of translation is due to several factors, one of which is the absence of molecular tools for the robust quantification of ligand density on the surface of liposomes. We report here for the first time the quantification of proteins attached to the surface of small unilamellar liposomes using single-molecule fluorescence imaging. Liposomes were surface-functionalized with fluorescently labeled human proteins previously validated to target the cancer cell surface biomarkers plasminogen activator inhibitor-2 (PAI-2) and trastuzumab (TZ, Herceptin®). These protein-conjugated liposomes were visualized using a custom-built wide-field fluorescence microscope with single-molecule sensitivity. By counting the photobleaching steps of the fluorescently labeled proteins, we calculated the number of attached proteins per liposome, which was 11 ± 4 proteins for single-ligand liposomes. Imaging of dual-ligand liposomes revealed stoichiometries of the two attached proteins in accordance with the molar ratios of protein added during preparation. Preparation of PAI-2/TZ dual-ligand liposomes via two different methods revealed that the post-insertion method generated liposomes with a more equal representation of the two differently sized proteins, demonstrating the ability of this preparation method to enable better control of liposome protein densities. We conclude that the single-molecule imaging method presented here is an accurate and reliable quantification tool for determining ligand density and stoichiometry on the surface of liposomes. This method has the potential to allow for comprehensive characterization of novel ligand-directed liposomes that should facilitate the translation of these nanotherapies through to the clinic.

Real-time single-molecule observation of rolling-circle DNA replication

Publisher: Oxford University Press (OUP)

Date: 20-01-2009

DOI: 10.1093/NAR/GKP006

Single-molecule fluorescence-based approach reveals novel mechanistic insights into human small heat shock protein chaperone function

Publisher: Elsevier BV

Date: 2021

DOI: 10.1074/JBC.RA120.015419

Highly-parallel microfluidics-based force spectroscopy on single cytoskeletal motors

Publisher: Cold Spring Harbor Laboratory

Date: 12-08-2020

DOI: 10.1101/2020.08.11.245910

Abstract: Cytoskeletal motors transform chemical energy into mechanical work to drive essential cellular functions. Optical trapping experiments have provided crucial insights into the operation of these molecular machines under load. However, the throughput of such force spectroscopy experiments is typically limited to one measurement at a time. Here, we introduce a highly-parallel, microfluidics-based method that allows for rapid collection of force-dependent motility parameters of cytoskeletal motors with two orders of magnitude improvement in throughput compared to currently available methods. We apply tunable hydrodynamic forces to stepping kinesin-1 motors via DNA-tethered beads and utilize a large field of view to simultaneously track the velocities, run lengths and interaction times of hundreds of in idual kinesin-1 molecules under varying resisting and assisting loads. Importantly, the 16-μm long DNA tethers between the motors and the beads significantly reduces the vertical component of the applied force pulling the motors away from the microtubule. Our approach is readily applicable to other molecular systems and constitutes a new methodology for parallelized single-molecule force studies on cytoskeletal motors.

DNA is a co-factor for its own replication in Xenopus egg extracts

Publisher: Oxford University Press (OUP)

Date: 21-09-2011

DOI: 10.1093/NAR/GKQ739

Single-component reflecting objective for low-temperature spectroscopy in the entire visible region

Publisher: AIP Publishing

Date: 30-07-2007

DOI: 10.1063/1.2767778

Abstract: A single-component reflecting objective was constructed for low-temperature spectroscopy with optimal imaging and transmission properties at all visible wavelengths. The performance of the objective immersed in superfluid helium at a temperature of 1.5K was tested by comparing dark-field images of uncolored polymer beads taken at wavelengths of 400 and 800nm. Under conditions optimized for imaging at both wavelengths, the size of the image is & .3 times of the diffraction limit. The objective collects emission from a point source at focus with a solid angle of 0.32πsr.

A mechanism for the extension and unfolding of parallel telomeric G-quadruplexes by human telomerase at single-molecule resolution

Publisher: eLife Sciences Publications, Ltd

Date: 29-07-2020

DOI: 10.7554/ELIFE.56428

Abstract: Telomeric G-quadruplexes (G4) were long believed to form a protective structure at telomeres, preventing their extension by the ribonucleoprotein telomerase. Contrary to this belief, we have previously demonstrated that parallel-stranded conformations of telomeric G4 can be extended by human and ciliate telomerase. However, a mechanistic understanding of the interaction of telomerase with structured DNA remained elusive. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) microscopy and bulk-phase enzymology to propose a mechanism for the resolution and extension of parallel G4 by telomerase. Binding is initiated by the RNA template of telomerase interacting with the G-quadruplex nucleotide addition then proceeds to the end of the RNA template. It is only through the large conformational change of translocation following synthesis that the G-quadruplex structure is completely unfolded to a linear product. Surprisingly, parallel G4 stabilization with either small molecule ligands or by chemical modification does not always inhibit G4 unfolding and extension by telomerase. These data reveal that telomerase is a parallel G-quadruplex resolvase.

Single-molecule fluorescence autocorrelation experiments on pentacene: The dependence of intersystem crossing on isotopic composition

Publisher: AIP Publishing

Date: 08-05-1999

DOI: 10.1063/1.478837

Abstract: Single pentacene molecules containing C13 or H1 in a pentacene-d14 doped p-terphenyl crystal have been studied by fluorescence autocorrelation. The triplet dynamics has been analyzed and a systematic dependence of the S1→T1 intersystem crossing rate on isotopic composition was found. This variation is discussed in terms of a modulation of the near resonance of the first excited singlet state S1 and vibrational levels of an intermediating triplet state T3 which results from the distinct isotope dependence of the zero-point energy of both electronic states.

Defects in DNA double-strand break repair re-sensitise antibiotic-resistant Escherichia coli to multiple bactericidal antibiotics

Publisher: Cold Spring Harbor Laboratory

Date: 25-01-2022

DOI: 10.1101/2022.01.24.477632

Abstract: Antibiotic resistance is becoming increasingly prevalent amongst bacterial pathogens and there is an urgent need to develop new types of antibiotics with novel modes of action. One promising strategy is to develop resistance-breaker compounds, which inhibit resistance mechanisms and thus re-sensitise bacteria to existing antibiotics. In the current study, we identify bacterial DNA double-strand break repair as a promising target for the development of resistance-breaking co-therapies. We examined genetic variants of Escherichia coli that combined antibiotic-resistance determinants with DNA repair defects. We observed that defects in the double-strand break repair pathway led to significant re-sensitisation towards five bactericidal antibiotics representing different functional classes. Effects ranged from partial to full re-sensitisation. For ciprofloxacin and nitrofurantoin, sensitisation manifested as a reduction in the minimum inhibitory concentration. For kanamycin and trimethoprim, sensitivity manifested through increased rates of killing at high antibiotic concentrations. For icillin, repair defects dramatically reduced antibiotic tolerance. Ciprofloxacin, nitrofurantoin, and trimethoprim induce the pro-mutagenic SOS response. Disruption of double-strand break repair strongly d ened the induction of SOS by these antibiotics. Our findings suggest that if break-repair inhibitors can be developed they could re-sensitise antibiotic-resistant bacteria to multiple classes of existing antibiotics and may supress the development of de novo antibiotic-resistance mutations.

Visualization of Membrane Fusion, One Particle at a Time

Publisher: American Chemical Society (ACS)

Date: 19-02-2013

DOI: 10.1021/BI301573W

Abstract: Protein-mediated fusion between phospholipid bilayers is a fundamental and necessary mechanism for many cellular processes. The short-lived nature of the intermediate states visited during fusion makes it challenging to capture precise kinetic information using classical, ensemble-averaging biophysical techniques. Recently, a number of single-particle fluorescence microscopy-based assays that allow researchers to obtain highly quantitative data about the fusion process by observing in idual fusion events in real time have been developed. These assays depend upon changes in the acquired fluorescence signal to provide a direct readout for transitions between the various fusion intermediates. The resulting data yield meaningful and detailed kinetic information about the transitory states en route to productive membrane fusion. In this review, we highlight recent in vitro and in vivo studies of membrane fusion at the single-particle level in the contexts of viral membrane fusion and SNARE-mediated synaptic vesicle fusion. These studies afford insight into mechanisms of coordination between fusion-mediating proteins as well as coordination of the overall fusion process with other cellular processes. The development of single-particle approaches to investigate membrane fusion and their successful application to a number of model systems have resulted in a new experimental paradigm and open up considerable opportunities to extend these methods to other biological processes that involve membrane fusion.

Coupling dTTP hydrolysis with DNA unwinding by the DNA helicase of bacteriophage T7.

Publisher: Elsevier BV

Date: 09-2011

DOI: 10.1074/JBC.M111.283796

Rapid single-molecule characterisation of nucleic-acid enzymes

Publisher: Cold Spring Harbor Laboratory

Date: 03-03-2022

DOI: 10.1101/2022.03.03.482895

Abstract: The activity of enzymes is traditionally characterised through bulk-phase biochemical methods that only report on population averages. Single-molecule methods are advantageous in elucidating kinetic and population heterogeneity but are often complicated, time consuming, and lacking statistical power. We present a highly generalisable and high-throughput single-molecule assay to rapidly characterise proteins involved in DNA metabolism. The assay exclusively relies on changes in total fluorescence intensity of surface-immobilised DNA templates as a result of DNA synthesis, unwinding or digestion. Combined with an automated data-analysis pipeline, our method provides enzymatic activity data of thousands of molecules in less than an hour. We demonstrate our method by characterising three fundamentally different nucleic-acid enzyme activities: digestion by the phage λ exonuclease, synthesis by the phage Phi29 polymerase, and unwinding by the E. coli UvrD helicase. We observe a previously unknown activity of the UvrD helicase to remove proteins tightly bound to the ends of DNA.

Nanoparticles for super-resolution microscopy and single-molecule tracking

Publisher: Springer Science and Business Media LLC

Date: 28-05-2018

DOI: 10.1038/S41592-018-0012-4

Abstract: We review the use of luminescent nanoparticles in super-resolution imaging and single-molecule tracking, and showcase novel approaches to super-resolution imaging that leverage the brightness, stability, and unique optical-switching properties of these nanoparticles. We also discuss the challenges associated with their use in biological systems, including intracellular delivery and molecular targeting. In doing so, we hope to provide practical guidance for biologists and continue to bridge the fields of super-resolution imaging and nanoparticle engineering to support their mutual advancement.

Single-molecule binding experiments on long time scales

Publisher: AIP Publishing

Date: 08-2010

DOI: 10.1063/1.3473936

Abstract: We describe an approach for performing single-molecule binding experiments on time scales from hours to days, allowing for the observation of slower kinetics than have been previously investigated by single-molecule techniques. Total internal reflection fluorescence microscopy is used to image the binding of labeled ligand to molecules specifically coupled to the surface of an optically transparent flow cell. Long-duration experiments are enabled by ensuring sufficient positional, chemical, thermal, and image stability. Principal components of this experimental stability include illumination timing, solution replacement, and chemical treatment of solution to reduce photodamage and photobleaching and autofocusing to correct for spatial drift.

Pyrovanadolysis, a pyrophosphorolysis-like reaction mediated by pyrovanadate, Mn2+, and DNA polymerase of bacteriophage T7

Publisher: Elsevier BV

Date: 08-2011

DOI: 10.1074/JBC.M111.250944

Single-molecule visualization of conformational changes and substrate transport in the vitamin B12 ABC importer BtuCD-F

Publisher: Springer Science and Business Media LLC

Date: 21-11-2017

DOI: 10.1038/S41467-017-01815-7

Abstract: ATP-binding cassette (ABC) transporters form the largest class of active membrane transport proteins. Binding and hydrolysis of ATP by their highly conserved nucleotide-binding domains drive conformational changes of the complex that mediate transport of substrate across the membrane. The vitamin B 12 importer BtuCD-F in Escherichia coli is an extensively studied model system. The periplasmic soluble binding protein BtuF binds the ligand the transmembrane and ATPase domains BtuCD mediate translocation. Here we report the direct observation at the single-molecule level of ATP, vitamin B 12 and BtuF-induced events in the transporter complex embedded in liposomes. Single-molecule fluorescence imaging techniques reveal that membrane-embedded BtuCD forms a stable complex with BtuF, regardless of the presence of ATP and vitamin B 12 . We observe that a vitamin B 12 molecule remains bound to the complex for tens of seconds, during which several ATP hydrolysis cycles can take place, before it is being transported across the membrane.

Probing molecular choreography through single-molecule biochemistry

Publisher: Springer Science and Business Media LLC

Date: 12-2015

DOI: 10.1038/NSMB.3119

Abstract: Single-molecule approaches are having a dramatic impact on views of how proteins work. The ability to observe molecular properties at the single-molecule level allows characterization of subpopulations and acquisition of detailed kinetic information that would otherwise be hidden in the averaging over an ensemble of molecules. In this Perspective, we discuss how such approaches have successfully been applied to in vitro-reconstituted systems of increasing complexity.

iSBatch: a batch-processing platform for data analysis and exploration of live-cell single-molecule microscopy images and other hierarchical datasets

Publisher: Royal Society of Chemistry (RSC)

Date: 2015

DOI: 10.1039/C5MB00321K

Abstract: iSBatch: an ImageJ plugin for fast evaluation of analysis pipelines and visual exploration of datasets.

Droplet microfluidics for kinetic studies of viral fusion

Publisher: AIP Publishing

Date: 03-2016

DOI: 10.1063/1.4943126

Abstract: Viral infections remain a major threat to public health. The speed with which viruses are evolving drug-resistant mutations necessitates the further development of antiviral therapies with a large emphasis on drug discovery. To facilitate these efforts, there is a need for robust, high-throughput assays that allow the screening of large libraries of compounds, while enabling access to detailed kinetic data on their antiviral activity. We report here the development of a droplet-based microfluidic platform to probe viral fusion, an early critical step in infection by membrane-enveloped viruses such as HIV, Hepatitis C, and influenza. Using influenza A, we demonstrate the measurement of the kinetics of fusion of virions with target liposomes with sub-second temporal resolution. In analogy with acidification of the endosome that triggers fusion in a cellular context, we acidify the content of aqueous droplets containing virions and liposomes in situ by introducing acid from the dispersed phase and visualize the kinetics of fusion by using fluorescent probes.

The drivers of antibiotic use and misuse: the development and investigation of a theory driven community measure

Publisher: Springer Science and Business Media LLC

Date: 30-10-2019

DOI: 10.1186/S12889-019-7796-8

Abstract: Antimicrobial resistance is a global public health concern, with extensive associated health and economic implications. Actions to slow and contain the development of resistance are imperative. Despite the fact that overuse and misuse of antibiotics are highlighted as major contributing factors to this resistance, no sufficiently validated measures aiming to investigate the drivers behind consumer behaviour amongst the general population are available. The objective of this study was to develop and investigate the psychometric properties of an original, novel and multiple-item questionnaire, informed by the Theory of Planned Behaviour, to measure factors contributing to self-reported antibiotic use within the community. A three-phase process was employed, including literature review and item generation expert panel review and pre-test. Investigation of the questionnaire was subsequently conducted through a cross-sectional, anonymous survey. Orthogonal principal analysis with varimax rotation, cronbach alpha and linear mixed-effects modelling analyses were conducted. A 60 item questionnaire was produced encompassing demographics, social desirability, three constructs of the Theory of Planned Behaviour including: attitudes and beliefs subjective norm perceived behavioural control behaviour and a covariate – knowledge. Three hundred seventy-three participants completed the survey. Eighty participants (21%) were excluded due to social desirability concerns, with data from the remaining 293 participants analysed. Results showed modest but acceptable levels of internal reliability, with high inter-item correlations within each construct. All four variables and the outcome variable of antibiotic use behaviour comprised four items with the exception of social norms, for which there were two items, producing a final 18 item questionnaire. Perceived behavioural control, social norms, the interaction between attitudes and beliefs and knowledge, and the presence of a healthcare worker in the family were all significant predictors of antibiotic use behaviour. All other predictors tested produced a nonsignificant relationship with the outcome variable of self-reported antibiotic use. This study successfully developed and validated a novel tool which assesses factors influencing community antibiotic use and misuse. The questionnaire can be used to guide appropriate intervention strategies to reduce antibiotic misuse in the general population. Future research is required to assess the extent to which this tool can guide community-based intervention strategies.

Multi-year antimicrobial-resistance trends in urine Escherichia coli isolates from both community-based and hospital-based laboratories of an Australian local health district

Publisher: Elsevier BV

Date: 12-2022

DOI: 10.1016/J.JGAR.2022.11.008

Abstract: Efforts to monitor and combat antimicrobial resistance (AMR) are typically focused on the hospital-based laboratory setting. The aim of this study was to longitudinally examine and compare trends in AMR among urine Escherichia coli isolates from a private community-based laboratory and a public hospital-based laboratory in an Australian local health district. A total of 108 262 urine E. coli isolates from a public hospital-based laboratory (N = 34 103) and a private community-based laboratory (N = 74 159) in a single health district between 2007-2019 were analysed. Linear regression was used to identify significance of change in AMR rates in both laboratories independently and detect any significant interaction of each setting in proportional change over the study period. Similar AMR trends were detected among urinary E. coli isolates in private community-based laboratory and public hospital-based laboratory settings over 12 y. AMR rates were consistently higher in the public hospital-based setting. Ampicillin was the only antibiotic for which the E. coli resistance trend did not significantly change over the time period in either laboratory setting. All other antibiotics showed a significant increase in AMR rates over time in both settings. AMR rates in both the private community-based laboratory and public hospital-based laboratory settings increased over time and were consistently higher in the public hospital-based laboratory setting. Since private laboratories handle the vast majority of pathology volumes in community outpatient settings in Australia, interventions incorporating the community-based laboratory setting are critical to addressing AMR in the community.

Lipids Activate SecA for High Affinity Binding to the SecYEG Complex

Publisher: Elsevier BV

Date: 10-2016

DOI: 10.1074/JBC.M116.743831

ParB spreading requires DNA bridging

Publisher: Cold Spring Harbor Laboratory

Date: 14-05-2014

DOI: 10.1101/GAD.242206.114

Abstract: The parABS system is a widely employed mechanism for plasmid partitioning and chromosome segregation in bacteria. ParB binds to parS sites on plasmids and chromosomes and associates with broad regions of adjacent DNA, a phenomenon known as spreading. Although essential for ParB function, the mechanism of spreading remains poorly understood. Using single-molecule approaches, we discovered that Bacillus subtilis ParB (Spo0J) is able to trap DNA loops. Point mutants in Spo0J that disrupt DNA bridging are defective in spreading and recruitment of structural maintenance of chromosomes (SMC) condensin complexes in vivo. DNA bridging helps to explain how a limited number of Spo0J molecules per parS site (∼20) can spread over many kilobases and suggests a mechanism by which ParB proteins could facilitate the loading of SMC complexes. We show that DNA bridging is a property of erse ParB homologs, suggesting broad evolutionary conservation.

Bisecting Microfluidic Channels with Metallic Nanowires Fabricated by Nanoskiving

Publisher: American Chemical Society (ACS)

Date: 05-02-2016

DOI: 10.1021/ACSNANO.5B07996

Abstract: This paper describes the fabrication of millimeter-long gold nanowires that bisect the center of microfluidic channels. We fabricated the nanowires by nanoskiving and then suspended them over a trench in a glass structure. The channel was sealed by bonding it to a complementary poly(dimethylsiloxane) structure. The resulting structures place the nanowires in the region of highest flow, as opposed to the walls, where it approaches zero, and expose their entire surface area to fluid. We demonstrate active functionality, by constructing a hot-wire anemometer to measure flow through determining the change in resistance of the nanowire as a function of heat dissipation at low voltage (<5 V). Further, passive functionality is demonstrated by visualizing in idual, fluorescently labeled DNA molecules attached to the wires. We measure rates of flow and show that, compared to surface-bound DNA strands, elongation saturates at lower rates of flow and background fluorescence from nonspecific binding is reduced.

Mechanism of transcription modulation by the transcription-repair coupling factor

Publisher: Oxford University Press (OUP)

Date: 31-05-2022

DOI: 10.1093/NAR/GKAC449

Abstract: Elongation by RNA polymerase is dynamically modulated by accessory factors. The transcription-repair coupling factor (TRCF) recognizes paused/stalled RNAPs and either rescues transcription or initiates transcription termination. Precisely how TRCFs choose to execute either outcome remains unclear. With Escherichia coli as a model, we used single-molecule assays to study dynamic modulation of elongation by Mfd, the bacterial TRCF. We found that nucleotide-bound Mfd converts the elongation complex (EC) into a catalytically poised state, presenting the EC with an opportunity to restart transcription. After long-lived residence in this catalytically poised state, ATP hydrolysis by Mfd remodels the EC through an irreversible process leading to loss of the RNA transcript. Further, biophysical studies revealed that the motor domain of Mfd binds and partially melts DNA containing a template strand overhang. The results explain pathway choice determining the fate of the EC and provide a molecular mechanism for transcription modulation by TRCF.

When proteins play tag: the dynamic nature of the replisome

Publisher: Springer Science and Business Media LLC

Date: 04-07-2019

DOI: 10.1007/S12551-019-00569-4

Regulation of Mutagenic DNA Polymerase V Activation in Space and Time

Publisher: Public Library of Science (PLoS)

Date: 28-08-2015

DOI: 10.1371/JOURNAL.PGEN.1005482

A general approach to break the concentration barrier in single-molecule imaging

Publisher: Springer Science and Business Media LLC

Date: 09-09-2012

DOI: 10.1038/NMETH.2174

University Of Sydney

Location: No location found

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Harvard Medical School

Location: United States of America

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Harvard University

Location: United States of America

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Rijksuniversiteit Groningen

Location: Netherlands

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Universiteit Leiden

Location: Netherlands

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University Of Sydney

Location: Australia

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University Of Wollongong

Location: Australia

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Superresolution Fluorescence Imaging In Microbiology

Start Date: 2016

End Date: 2016

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE210100042

Start Date: 2021

End Date: 12-2022

Amount: $970,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0877658

Start Date: 05-2008

End Date: 12-2013

Amount: $1,098,934.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP180100858

Start Date: 2018

End Date: 12-2020

Amount: $524,298.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP210100167

Start Date: 2021

End Date: 04-2024

Amount: $600,364.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP150100956

Start Date: 2015

End Date: 12-2017

Amount: $778,791.00

Funder: Australian Research Council

Linkage - International - Grant ID: LX0882706

Start Date: 2008

End Date: 12-2012

Amount: $37,000.00

Funder: Australian Research Council

Australian Laureate Fellowships - Grant ID: FL140100027

Start Date: 04-2015

End Date: 12-2020

Amount: $2,898,150.00

Funder: Australian Research Council

Linkage - International - Grant ID: LX0668812

Start Date: 06-2006

End Date: 12-2008

Amount: $20,400.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP220103466

Start Date: 2022

End Date: 12-2025

Amount: $537,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE160100127

Start Date: 2016

End Date: 12-2016

Amount: $355,000.00

Funder: Australian Research Council

Industrial Transformation Training Centres - Grant ID: IC200100052

Start Date: 03-2021

End Date: 03-2026

Amount: $4,789,838.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE150100163

Start Date: 2015

End Date: 12-2016

Amount: $560,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE210100166

Start Date: 2021

End Date: 12-2021

Amount: $626,800.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE190100130

Start Date: 2019

End Date: 12-2019

Amount: $1,480,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE180100043

Start Date: 2018

End Date: 12-2019

Amount: $435,279.00

Funder: Australian Research Council

ARC Centres Of Excellence - Grant ID: CE230100021

Start Date: 12-2023

End Date: 12-2030

Amount: $35,000,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE180100001

Start Date: 2018

End Date: 12-2018

Amount: $345,475.00

Funder: Australian Research Council