Sortase-mediated fluorescent labeling of CRISPR complexes

Dillard, Kaylee E.; Schaub, Jeffrey M.; Brown, Maxwell W.; Saifuddin, Fatema A.; Xiao, Yibei; Hernandez, Erik T.; Dahlhauser, Samuel D.; Anslyn, Eric V.; Ke, Ailong; Finkelstein, Ilya J.

doi:10.1016/bs.mie.2018.10.031

Cited by 10 publications

(5 citation statements)

References 32 publications

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“…We explored a Sortase A-mediated labeling strategy to fluorescently label Tau protein while causing minimal perturbation at the core region of the protein in the fibrillar state. − The method relies on the addition of a specific recognition motif LPXTG (X being any amino acid) to the C terminus of recombinantly expressed Tau protein. Sortase A from Staphylococcus aureus cleaves off the terminal glycine and forms a thioester with threonine.…”

Section: Resultsmentioning

confidence: 99%

Site-Specific C-Terminal Fluorescent Labeling of Tau Protein

Bryan

Awasthi

et al. 2022

ACS Omega

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Formation of Tau protein aggregates in neurons is a pathological hallmark of several neurodegenerative diseases, including Alzheimer’s disease. Fluorescently labeled Tau protein is therefore useful to study the aggregation of these pathological proteins and to identify potential therapeutic targets. Conventionally, cysteine residues are used for labeling Tau proteins; however, the full-length Tau isoform contains two cysteine residues in the microtubule-binding region, which are implicated in Tau aggregation by forming intermolecular disulfide bonds. To prevent the fluorescent label from disturbing the microtubule binding region, we developed a strategy to fluorescently label Tau at its C-terminus while leaving cysteine residues unperturbed. We took advantage of a Sortase A-mediated transpeptidation approach to bind a short peptide (GGGH6-Alexa647) with a His-tag and a covalently attached Alexa 647 fluorophore to the C-terminus of Tau. This reaction relies on the presence of a Sortase recognition motif (LPXTG), which we attached to the C-terminus of recombinantly expressed Tau. We demonstrate that C-terminal modification of Tau protein results in no significant differences between the native and C-terminally labeled Tau monomer with regard to aggregation kinetics, secondary structure, and fibril morphology.

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Section: Resultsmentioning

confidence: 99%

Site-Specific C-Terminal Fluorescent Labeling of Tau Protein

Bryan

Awasthi

et al. 2022

ACS Omega

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“…We first assayed how Polθ-h counteracts RPA-coated ssDNA because RPA inhibits hybridization of heteroduplex oligos during TMEJ (19). We monitored the removal of fluorescent RPA-GFP because multiple fluorescent labeling strategies resulted in hypoactive Polθ-h ( Figure S2A ) (39). In this assay, ssDNA curtains are assembled with RPA-GFP.…”

Section: Resultsmentioning

confidence: 99%

Polymerase theta-helicase promotes end joining by stripping single-stranded DNA-binding proteins and bridging DNA ends

Soniat

Finkelstein

2021

Preprint

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Homologous recombination-deficient cancers rely on DNA polymerase Theta (Polθ)-Mediated End Joining (TMEJ), an alternative double-strand break repair pathway. Polθ is the only vertebrate polymerase that encodes an N-terminal superfamily 2 (SF2) helicase domain, but the role of this helicase domain in TMEJ remains unclear. Using single-molecule imaging, we demonstrate that Polθ-helicase (Polθ-h) is a highly processive single-stranded DNA (ssDNA) motor protein that can efficiently strip Replication Protein A (RPA) from ssDNA. Polθ-h also has a limited capacity for disassembling RAD51 filaments but is not processive on double- stranded DNA. Polθ-h can bridge two non-complementary DNA strands in trans. PARylation of Polθ-h by PARP-1 resolves these DNA bridges. We conclude that Polθ-h removes RPA and RAD51 filaments and mediates bridging of DNA overhangs to aid in polymerization by the Polθ polymerase domain.

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“…Single-tethered DNA curtains were also combined with biochemical assays to gather more molecular details about Abo1-mediated histone loading onto DNA and the ongoing interactions between Abo1 and DNA [112]. Another study combined single-tethered and double-tethered DNA curtains to monitor the motion of individual fluorescently labeled Type I-E CRISPR-Cas proteins on DNA [113]. It revealed that Cas1-Cas2 integrase is able to non-specifically sample the DNA through 3D collisions with a short lifetime, while Cas3 helicase/nuclease translocates on DNA by using two main modes, as at the target sequence it either moves in conjunction with the 11-subunit crRNA-containing Cascade surveillance complex or independently away from Cascade.…”

Section: Single-tethered Dna Curtainsmentioning

confidence: 99%

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

2022

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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.

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Sortase-mediated fluorescent labeling of CRISPR complexes

Cited by 10 publications

References 32 publications

Site-Specific C-Terminal Fluorescent Labeling of Tau Protein

Site-Specific C-Terminal Fluorescent Labeling of Tau Protein

Polymerase theta-helicase promotes end joining by stripping single-stranded DNA-binding proteins and bridging DNA ends

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

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