Implementing fluorescence enhancement, quenching, and FRET for investigating flap endonuclease 1 enzymatic reaction at the single-molecule level

Sobhy, Mohamed A.; Tehseen, Muhammad; Takahashi, Masateru; Bralić, Amer; Biasio, Alfredo De; Hamdan, Samir M.

doi:10.1016/j.csbj.2021.07.029

Cited by 10 publications

(9 citation statements)

References 112 publications

(233 reference statements)

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“…This was first demonstrated for sCy dyes by Steffen et al in the presence of different RNA structures (Figures 4(A), 5) [75]. The dependence of NAIFE on the chemical microenvironment can now be used to investigate the degree of folding of the nucleic acid or the interaction probability with binding partners, such as hybridising DNA fragments [86] or a recently developed DNA-aptamer sensor [87].…”

Section: Nucleic Acid-induced Fluorescence Enhancement (Naife)mentioning

confidence: 91%

A new twist on PIFE: photoisomerisation-related fluorescence enhancement

Ploetz,

Ambrose,

Barth

et al. 2023

Methods Appl. Fluoresc.

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PIFE was first used as an acronym for protein-induced fluorescence enhancement, which refers to the increase in fluorescence observed upon the interaction of a fluorophore, such as a cyanine, with a protein. This fluorescence enhancement is due to changes in the rate of cis/trans photoisomerisation. It is clear now that this mechanism is generally applicable to interactions with any biomolecule and, in this review, we propose that PIFE is thereby renamed according to its fundamental working principle as photoisomerisation-related fluorescence enhancement, keeping the PIFE acronym intact. We discuss the photochemistry of cyanine fluorophores, the mechanism of PIFE, its advantages and limitations, and recent approaches to turn PIFE into a quantitative assay. We provide an overview of its current applications to different biomolecules and discuss potential future uses, including the study of protein-protein interactions, protein-ligand interactions and conformational changes in biomolecules.

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Section: Nucleic Acid-induced Fluorescence Enhancement (Naife)mentioning

confidence: 91%

A new twist on PIFE: photoisomerisation-related fluorescence enhancement

Ploetz,

Ambrose,

Barth

et al. 2023

Methods Appl. Fluoresc.

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“…Additionally, several model systems of proteins and enzymes, such as endonucleases, polymerases, and lipases, involved in biological processes like DNA replication have been characterized with similar methods (i.e., single-molecule localization, confocal microscopy, and TIRF) to provide insight into kinetic mechanisms. 19,142,[213][214][215] As an example, Sobhy et al utilized FRET and confocal microscopy and found that flap endonuclease 1 (FEN1) blends DNA molecules in accordance with a diffusion-limited model. While DNA sequencing is not within the scope of this perspective, there are multiple studies and reviews discussing the applications of single-molecule techniques to image these processes.…”

Section: Biopolymer Systemsmentioning

confidence: 99%

Single‐molecule fluorescence microscopy for imaging chemical reactions: Recent progress and future opportunities for advancing polymer systems

Dunn,

Valdez,

Qiang

2023

Journal of Polymer Science

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Single‐molecule fluorescence (smFL) imaging techniques have evolved greatly over the past two decades to encompass the ability to monitor chemical reactions, providing unique advantages of non‐invasive sample preparation and characterization, labeling specificity, and high spatial and temporal resolutions. This work summarizes the recent progress in this important area by first providing a brief overview of different smFL techniques, including their common optical setups and working principles. We then introduce recent developments of smFL to characterize various model chemical reaction systems, such as biochemical synthesis, catalyzed systems, and nanomaterial assembly. Furthermore, several representative areas of using smFL to understand polymer reactions are discussed, including understanding interfacial phenomenon and polymerization kinetics, as well as characterizing electrochemical reactions. We also highlight the outlook of this exciting field and potential opportunities for further development and application of smFL to enable advances in polymer chemistry and physics.

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“…We then focused on substrate conformational requirements and handoff mechanisms among MOF proteins. We previously deciphered conformational states during the FEN1 catalytic cycle using single-molecule FRET 13,14,36,37 (Fig. 2A).…”

Section: Reconstitution Of the Human Mof Reactionmentioning

confidence: 99%

Mechanistic investigation of human maturation of Okazaki fragments reveals slow kinetics

et al. 2022

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The final steps of lagging strand synthesis induce maturation of Okazaki fragments via removal of the RNA primers and ligation. Iterative cycles between Polymerase δ (Polδ) and Flap endonuclease-1 (FEN1) remove the primer, with an intermediary nick structure generated for each cycle. Here, we show that human Polδ is inefficient in releasing the nick product from FEN1, resulting in non-processive and remarkably slow RNA removal. Ligase 1 (Lig1) can release the nick from FEN1 and actively drive the reaction toward ligation. These mechanisms are coordinated by PCNA, which encircles DNA, and dynamically recruits Polδ, FEN1, and Lig1 to compete for their substrates. Our findings call for investigating additional pathways that may accelerate RNA removal in human cells, such as RNA pre-removal by RNase Hs, which, as demonstrated herein, enhances the maturation rate ~10-fold. They also suggest that FEN1 may attenuate the various activities of Polδ during DNA repair and recombination.

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Implementing fluorescence enhancement, quenching, and FRET for investigating flap endonuclease 1 enzymatic reaction at the single-molecule level

Abstract: Graphical abstract

Cited by 10 publications

References 112 publications

A new twist on PIFE: photoisomerisation-related fluorescence enhancement

A new twist on PIFE: photoisomerisation-related fluorescence enhancement

Single‐molecule fluorescence microscopy for imaging chemical reactions: Recent progress and future opportunities for advancing polymer systems

Mechanistic investigation of human maturation of Okazaki fragments reveals slow kinetics

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