An in Vitro Sample Generation Pipeline for High-Throughput Single-Molecule FRET Based Screening of Proteins

The DNA helicase Large helicase-related (Lhr) is present throughout archaea, including in the Asgard and Nanoarchaea, and has homologues in bacteria and eukaryotes. It is thought to function in DNA repair but in a context that is not known. Our data show that archaeal Lhr preferentially targets DNA replication fork structures. In a genetic assay, expression of archaeal Lhr gave a phenotype identical to the replication-coupled DNA repair enzymes Hel308 and RecQ. Purified archaeal Lhr preferentially unwound model forked DNA substrates compared with DNA duplexes, flaps and Holliday junctions, and unwound them with directionality. Single-molecule FRET measurements showed that binding of Lhr to a DNA fork causes ATP-independent distortion and base-pair melting at, or close to, the fork branchpoint. ATP-dependent directional translocation of Lhr resulted in fork DNA unwinding through the ‘parental’ DNA strands. Interaction of Lhr with replication forks in vivo and in vitro suggests that it contributes to DNA repair at stalled or broken DNA replication.

Download Full-text

High throughput mechanobiology: Force modulation of ensemble biochemical and cell-based assays

10.1101/2020.05.05.065912 ◽

2020 ◽

Cited By ~ 1

Author(s):

Ália dos Santos ◽

Natalia Fili ◽

David S. Pearson ◽

Yukti Hari-Gupta ◽

Christopher P. Toseland

Keyword(s):

High Throughput ◽

Single Molecule ◽

Receptor Activation ◽

Magnetic Tweezers ◽

Live Cells ◽

Mechanical Stimuli ◽

Cellular Assays ◽

Novel Approach ◽

Microplate Reader

ABSTRACTMechanobiology is focused on how the physical forces and the mechanical properties of proteins, cells and tissues contribute to physiology and disease. While the response of proteins and cells to mechanical stimuli is critical for function, the tools to probe these activities are typically restricted to single molecule manipulations. Here, we have developed a novel microplate reader assay to encompass mechanical measurements with ensemble biochemical and cellular assays, using a microplate lid modified with magnets. This configuration enables multiple static magnetic tweezers to function simultaneously across the microplate, thereby greatly increasing throughput. The broad applicability and versatility of our approach has been demonstrated through in vitro force-induced enzymatic activity and conformation changes, along with force-induced receptor activation and their downstream signalling pathways in live cells. Overall, our methodology allows for the first-time ensemble biochemical and cell-based assays to be performed under force, in high throughput format. This novel approach would substantially add to the mechano-biological toolbox and increase the availability of mechanobiology measurements.

Download Full-text

DNA Flow-Stretch Assays for Studies of Protein-DNA Interactions at the Single-Molecule Level

Applied Nano ◽

10.3390/applnano3010002 ◽

2022 ◽

Vol 3 (1) ◽

pp. 16-41

Author(s):

Aurimas Kopūstas ◽

Mindaugas Zaremba ◽

Marijonas Tutkus

Keyword(s):

High Throughput ◽

Single Molecule ◽

Dna Interaction ◽

Dna Interactions ◽

Ensemble Averaging ◽

Target Search ◽

Single Molecule Level ◽

Protein Dna Interactions ◽

Long Time

Protein-DNA interactions are the core of the cell’s molecular machinery. For a long time, conventional biochemical methods served as a powerful investigatory basis of protein-DNA interactions and target search mechanisms. Currently single-molecule (SM) techniques have emerged as a complementary tool for studying these interactions and have revealed plenty of previously obscured mechanistic details. In comparison to the traditional ones, SM methods allow direct monitoring of individual biomolecules. Therefore, SM methods reveal reactions that are otherwise hidden by the ensemble averaging observed in conventional bulk-type methods. SM biophysical techniques employing various nanobiotechnology methods for immobilization of studied molecules grant the possibility to monitor individual reaction trajectories of biomolecules. Next-generation in vitro SM biophysics approaches enabling high-throughput studies are characterized by much greater complexity than the ones developed previously. Currently, several high-throughput DNA flow-stretch assays have been published and have shown many benefits for mechanistic target search studies of various DNA-binding proteins, such as CRISPR-Cas, Argonaute, various ATP-fueled helicases and translocases, and others. This review focuses on SM techniques employing surface-immobilized and relatively long DNA molecules for studying protein-DNA interaction mechanisms.

Download Full-text

Single-molecule fluorescence resonance energy transfer techniques on rotary ATP synthases

Biological Chemistry ◽

10.1515/bc.2011.001 ◽

2011 ◽

Vol 392 (1-2) ◽

Cited By ~ 13

Author(s):

Michael Börsch

Keyword(s):

Energy Transfer ◽

Fluorescence Resonance Energy Transfer ◽

Single Molecule ◽

Conformational Changes ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Single Molecule Fret ◽

Intensity Changes ◽

Fluorescence Resonance

Abstract Conformational changes of proteins can be monitored in real time by fluorescence resonance energy transfer (FRET). Two different fluorophores have to be attached to those protein domains which move during function. Distance fluctuations between the fluorophores are measured by relative fluorescence intensity changes or fluorescence lifetime changes. The rotary mechanics of the two motors of FoF1-ATP synthase have been studied in vitro by single-molecule FRET. The results are summarized and perspectives for other transport ATPases are discussed.

Download Full-text

Single-molecule FRET study of SNARE-mediated membrane fusion

Bioscience Reports ◽

10.1042/bsr20110011 ◽

2011 ◽

Vol 31 (6) ◽

pp. 457-463 ◽

Cited By ~ 14

Author(s):

Jiajie Diao ◽

Yuji Ishitsuka ◽

Woo-Ri Bae

Keyword(s):

Membrane Fusion ◽

Lipid Bilayers ◽

Single Molecule ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Cellular Processes ◽

Single Molecule Fret ◽

Protein Receptor ◽

Hydrophobic Cores

Membrane fusion is one of the most important cellular processes by which two initially distinct lipid bilayers merge their hydrophobic cores, resulting in one interconnected structure. Proteins, called SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor), play a central role in the fusion process that is also regulated by several accessory proteins. In order to study the SNARE-mediated membrane fusion, the in vitro protein reconstitution assay involving ensemble FRET (fluorescence resonance energy transfer) has been used over a decade. In this mini-review, we describe several single-molecule-based FRET approaches that have been applied to this field to overcome the shortage of the bulk assay in terms of protein and fusion dynamics.

Download Full-text

Multispot single-molecule FRET: High-throughput analysis of freely diffusing molecules

PLoS ONE ◽

10.1371/journal.pone.0175766 ◽

2017 ◽

Vol 12 (4) ◽

pp. e0175766 ◽

Cited By ~ 17

Author(s):

Antonino Ingargiola ◽

Eitan Lerner ◽

SangYoon Chung ◽

Francesco Panzeri ◽

Angelo Gulinatti ◽

...

Keyword(s):

High Throughput ◽

Single Molecule ◽

High Throughput Analysis ◽

Throughput Analysis ◽

Single Molecule Fret

Download Full-text

The yeast Hrq1 helicase stimulates Pso2 translesion nuclease activity and thereby promotes DNA interstrand crosslink repair

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013626 ◽

2020 ◽

Vol 295 (27) ◽

pp. 8945-8957 ◽

Cited By ~ 3

Author(s):

Cody M. Rogers ◽

Chun-Ying Lee ◽

Samuel Parkins ◽

Nicholas J. Buehler ◽

Sabine Wenzel ◽

...

Keyword(s):

Single Molecule ◽

Nuclease Activity ◽

Dna Lesions ◽

Principal Mechanism ◽

Single Molecule Fret ◽

Interstrand Crosslink ◽

Physical Barriers ◽

A Site ◽

Interstrand Crosslink Repair

DNA interstrand crosslink (ICL) repair requires a complex network of DNA damage response pathways. Removal of the ICL lesions is vital, as they are physical barriers to essential DNA processes that require the separation of duplex DNA, such as replication and transcription. The Fanconi anemia (FA) pathway is the principal mechanism for ICL repair in metazoans and is coupled to DNA replication. In Saccharomyces cerevisiae, a vestigial FA pathway is present, but ICLs are predominantly repaired by a pathway involving the Pso2 nuclease, which is hypothesized to use its exonuclease activity to digest through the lesion to provide access for translesion polymerases. However, Pso2 lacks translesion nuclease activity in vitro, and mechanistic details of this pathway are lacking, especially relative to FA. We recently identified the Hrq1 helicase, a homolog of the disease-linked enzyme RecQ-like helicase 4 (RECQL4), as a component of Pso2-mediated ICL repair. Here, using genetic, biochemical, and biophysical approaches, including single-molecule FRET (smFRET)– and gel-based nuclease assays, we show that Hrq1 stimulates the Pso2 nuclease through a mechanism that requires Hrq1 catalytic activity. Importantly, Hrq1 also stimulated Pso2 translesion nuclease activity through a site-specific ICL in vitro. We noted that stimulation of Pso2 nuclease activity is specific to eukaryotic RecQ4 subfamily helicases, and genetic and biochemical data suggest that Hrq1 likely interacts with Pso2 through their N-terminal domains. These results advance our understanding of FA-independent ICL repair and establish a role for the RecQ4 helicases in the repair of these detrimental DNA lesions.

Download Full-text

UvrD helicase activation by MutL involves rotation of its 2B subdomain

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1905513116 ◽

2019 ◽

Vol 116 (33) ◽

pp. 16320-16325 ◽

Cited By ~ 7

Author(s):

Yerdos A. Ordabayev ◽

Binh Nguyen ◽

Alexander G. Kozlov ◽

Haifeng Jia ◽

Timothy M. Lohman

Keyword(s):

Escherichia Coli ◽

Single Molecule ◽

Self Assembly ◽

Kinetic Studies ◽

Conformational State ◽

Helicase Activity ◽

Single Molecule Fret ◽

Open Conformation ◽

Dna Complex

Escherichia coli UvrD is a superfamily 1 helicase/translocase that functions in DNA repair, replication, and recombination. Although a UvrD monomer can translocate along single-stranded DNA, self-assembly or interaction with an accessory protein is needed to activate its helicase activity in vitro. Our previous studies have shown that an Escherichia coli MutL dimer can activate the UvrD monomer helicase in vitro, but the mechanism is not known. The UvrD 2B subdomain is regulatory and can exist in extreme rotational conformational states. By using single-molecule FRET approaches, we show that the 2B subdomain of a UvrD monomer bound to DNA exists in equilibrium between open and closed states, but predominantly in an open conformation. However, upon MutL binding to a UvrD monomer–DNA complex, a rotational conformational state is favored that is intermediate between the open and closed states. Parallel kinetic studies of MutL activation of the UvrD helicase and of MutL-dependent changes in the UvrD 2B subdomain show that the transition from an open to an intermediate 2B subdomain state is on the pathway to helicase activation. We further show that MutL is unable to activate the helicase activity of a chimeric UvrD containing the 2B subdomain of the structurally similar Rep helicase. Hence, MutL activation of the monomeric UvrD helicase is regulated specifically by its 2B subdomain.

Download Full-text

ENDO-Pore: High-throughput linked-end mapping of single DNA cleavage events using nanopore sequencing

10.1101/2021.07.02.450912 ◽

2021 ◽

Author(s):

Oscar E Torres Montaguth ◽

Stephen J Cross ◽

Kincaid W.A. Ingram ◽

Laura Lee ◽

Fiona M Diffin ◽

...

Keyword(s):

High Throughput ◽

Single Molecule ◽

Dna Cleavage ◽

Cleavage Site ◽

Rolling Circle Amplification ◽

Time Resolved ◽

Rolling Circle ◽

Precise Position ◽

And Function

Mapping the precise position of DNA cleavage events plays a key role in determining the mechanism and function of endonucleases. ENDO-Pore is a high-throughput nanopore-based method that allows the time resolved mapping single molecule DNA cleavage events in vitro. Following linearisation of a circular DNA substrate by the endonuclease, a resistance cassette is ligated recording the position of the cleavage event. A library of single cleavage events is constructed and subjected to rolling circle amplification to generate concatemers. These are sequenced and used to produce accurate consensus sequences. To identify the cleavage site(s), we developed CSI (Cleavage Site Investigator). CSI recognizes the ends of the cassette ligated into the cleaved substrate and triangulates the position of the dsDNA break. We firstly benchmarked ENDO-Pore using Type II restriction endonucleases. Secondly, we analysed the effect of crRNA length on the cleavage pattern of CRISPR Cas12a. Finally, we mapped the time-resolved DNA cleavage by the Type ISP restriction endonuclease LlaGI that introduces random double-strand breaks into its DNA substrates.

Download Full-text