Single-molecule characterization of compressed RecA nucleoprotein filaments

Alekseev, Aleksandr; Morozova, Natalia; Vedyaykin, Alexey; Yakimov, Alexander; Khodorkovskii, M. A.; Pobegalov, Georgii

doi:10.1016/j.bbrc.2022.04.130

Cited by 5 publications

(3 citation statements)

References 31 publications

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“…Optical trapping experiments were performed to analyze SMC protein binding to individual double-stranded and single-stranded DNA molecules. DNA manipulation was carried out inside a five-channel microfluidic system (uFlux, Lumicks), using two polystyrene microspheres coated with streptavidin (Spherotech, 2.1 μm diameter) and held by two optical traps according to the method presented in [20, 21]. A 11070 bp long linear DNA molecule (prlC) was used as substrate.…”

Section: Methodsmentioning

confidence: 99%

Properties of theUreaplasma parvumSMC protein related to its interaction with DNA

Rumyantseva,

Shutov,

Belenkaia

et al. 2024

Preprint

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SMC (Structural Maintenance of Chromosomes) ATPase proteins are a component of complexes of the same name that participate in the spatial organization of DNA in all living organisms - in bacteria, archaea and eukaryotes. In bacteria, SMC complexes perform several functions, among which the function related to DNA condensation and segregation is the most studied. SMC complexes in bacteria are thought to extrude loops and thus compact DNA, as well as promote the separation of daughter nucleoids. In this work, we determined some properties of the SMC ATPase of Ureaplasma parvum, a member of the Mollicutes class with a reduced genome. The results indicate that the properties of this protein differ from those of the well-studied ortholog from Bacillus subtilis, in particular, U. parvum SMC binds to double-stranded DNA rather than single-stranded DNA. This protein has also been shown to compact DNA under in vitro conditions. The ATPase activity of U. parvum SMC was estimated and shown to be induced by DNA binding to SMC. The results of current paper indicate that U. parvum SMC cross-links DNA even in the absence of ATP.

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Section: Methodsmentioning

confidence: 99%

Properties of theUreaplasma parvumSMC protein related to its interaction with DNA

Rumyantseva,

Shutov,

Belenkaia

et al. 2024

Preprint

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“…Optical trapping experiments were performed to visualize RecN::mCherry protein binding to individual double-stranded and single-stranded DNA molecules. DNA manipulation was carried out inside a five-channel microfluidic system (uFlux, Lumicks), using two polystyrene microspheres coated with streptavidin (Spherotech, 2.1 μm diameter) and held by two optical traps according to the method presented in [16,17]. Double-stranded linear DNA molecules ~23 kb length with biotinylated ends and obtained by modifying the DNA of bacteriophage lambda were initially used as a DNA substrate.…”

Section: Visualization Of Recn::mcherry Dna Interaction Dynamics At T...mentioning

confidence: 99%

Features of the DNAEscherichia coliRecN interaction revealed by fluorescence microscopy and single-molecule methods

Roshektaeva,

Alekseev,

Vedyaykin

et al. 2024

Preprint

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The SOS response is a condition that occurs in bacterial cells after DNA damage. In this state, the bacterium is able to recover the integrity of its genome. Due to the increased level of mutagenesis in cells during the repair of DNA double-strand breaks, the SOS response is also an important mechanism for bacterial adaptation to the antibiotics. One of the key proteins of the SOS response is the SMC-like protein RecN, which helps the RecA recombinase to find a homologous DNA template for repair. In this work, the localization of the recombinant RecN protein in livingEscherichia colicells was revealed using fluorescence microscopy. It has been shown that the RecN, outside the SOS response, is predominantly localized at the poles of the cell, and in dividing cells, also localized at the center. Usingin vitromethods including fluorescence microscopy and optical tweezers, we show that RecN predominantly binds single-stranded DNA in an ATP-dependent manner. RecN has both intrinsic and single-stranded DNA-stimulated ATPase activity. The results of this work may be useful for better understanding of the SOS response mechanism and homologous recombination process.

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“…For single-molecule manipulations a custom-built dual trap optical tweezers setup based on the Axioimager.Z1 microscope was used as described previously [15,16]. In brief, two independent optical traps were generated using a Nd:YVO4 1064 nm CW laser (5 W, Spectra Physics BL-106C) and an oil immersion lens with a high numerical aperture.…”

Section: Optical Tweezers Setupmentioning

confidence: 99%

Catalytically inactive dKbCas12d guided by sgRNA and new insights into its binding through a single-molecule approach

Alekseev,

Klimko,

Abramova

et al. 2023

Preprint

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CRISPR-Cas12d is a distinct V-D type system discovered in the metagenomes of Candidate Phyla Radiation bacteria. It stands out from most closely related systems due to its 17-19 nucleotide short spacer region and specialized stabilizing scoutRNAs. We made significant improvements to this system by modifying its scoutRNA to create sgRNA, which greatly simplifies its use. We found mutations in the RuvC domain of the effector protein KbCas12d that resulted in loss of nuclease activity. We obtained two catalytically inactive dKbCas12d variants: D827A and E913A. Using the optical tweezers technique, we demonstrated the high specificity of dKbCas12d in binding targets on individual DNA molecules. Engineered sgRNA and catalytically inactive dKbCas12d variants have promising applications in biotechnology for the precise regulation of gene expression and molecular diagnostics.

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Single-molecule characterization of compressed RecA nucleoprotein filaments

Cited by 5 publications

References 31 publications

Properties of theUreaplasma parvumSMC protein related to its interaction with DNA

Properties of theUreaplasma parvumSMC protein related to its interaction with DNA

Features of the DNAEscherichia coliRecN interaction revealed by fluorescence microscopy and single-molecule methods

Catalytically inactive dKbCas12d guided by sgRNA and new insights into its binding through a single-molecule approach

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