Investigation of site-specific DNA binding with nicking endonuclease Nt.BspD6I at single molecule level by atomic force microscopy

Мачулин, А. В.; Deryusheva, Evgenia I.; Yunusova, A. K.; Zheleznaya, L. A.; Serdyuk, Igor N.

doi:10.1134/s0006350912030128

Cited by 4 publications

(4 citation statements)

References 9 publications

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“…Purified plasmid pET28b looked like a native and strongly supercoiled molecule ( Fig 5A ). It was digested by the site-specific nickase Nt.BspD6I, which recognized nine GAGTC sequences and made single stranded breaks in the top strand four bases towards the 3’-end [ 50 ]. As a result, the plasmid became relaxed and even fragmented ( Fig 5B ) since several binding sites are closely located in this DNA.…”

Section: Resultsmentioning

confidence: 99%

Modes of Escherichia coli Dps Interaction with DNA as Revealed by Atomic Force Microscopy

et al. 2015

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Multifunctional protein Dps plays an important role in iron assimilation and a crucial role in bacterial genome packaging. Its monomers form dodecameric spherical particles accumulating ~400 molecules of oxidized iron ions within the protein cavity and applying a flexible N-terminal ends of each subunit for interaction with DNA. Deposition of iron is a well-studied process by which cells remove toxic Fe2+ ions from the genetic material and store them in an easily accessible form. However, the mode of interaction with linear DNA remained mysterious and binary complexes with Dps have not been characterized so far. It is widely believed that Dps binds DNA without any sequence or structural preferences but several lines of evidence have demonstrated its ability to differentiate gene expression, which assumes certain specificity. Here we show that Dps has a different affinity for the two DNA fragments taken from the dps gene regulatory region. We found by atomic force microscopy that Dps predominantly occupies thermodynamically unstable ends of linear double-stranded DNA fragments and has high affinity to the central part of the branched DNA molecule self-assembled from three single-stranded oligonucleotides. It was proposed that Dps prefers binding to those regions in DNA that provide more contact pads for the triad of its DNA-binding bundle associated with one vertex of the protein globule. To our knowledge, this is the first study revealed the nucleoid protein with an affinity to branched DNA typical for genomic regions with direct and inverted repeats. As a ubiquitous feature of bacterial and eukaryotic genomes, such structural elements should be of particular care, but the protein system evolutionarily adapted for this function is not yet known, and we suggest Dps as a putative component of this system.

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Section: Resultsmentioning

confidence: 99%

Modes of Escherichia coli Dps Interaction with DNA as Revealed by Atomic Force Microscopy

et al. 2015

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“…Our result is contradictional to the proposed model of R.BspD6I complexed with DNA [14]; where the DNA is in a straight conformation. It seems that this model requires some correction considering the obtained data, particularly as also Machulin et al [28] observed a DNA bending by Nt.BspD6I using AFM.…”

Section: Estimation Of Dna Bending Angle Induced By Ntbspd6imentioning

confidence: 88%

Peculiarities of the interaction of the restriction endonuclease BspD6I with DNA containing its recognition site

Abrosimova

Кубарева

Migur

et al. 2016

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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“…The authors of ref. [9], using atomic force microscopy, confirmed the location of the enzyme at a specific site after its binding to DNA duplexes with a length of 920 bp adsorbed on a mica layer in the presence of Mg 2+ ions. In our group, using the circular permutation method based on differences in the mobility of DNA-protein complexes during polyacrylamide gel electrophoresis (PAGE), Nt.BspD6I has been shown to induce DNA bending by an angle of 66 • ± 4 • during complex formation and to be able to form a complex with the product of nicking [10].…”

Section: Introductionmentioning

confidence: 74%

Kinetic Analysis of the Interaction of Nicking Endonuclease BspD6I with DNA

et al. 2021

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Nicking endonucleases (NEs) are enzymes that incise only one strand of the duplex to produce a DNA molecule that is ‘nicked’ rather than cleaved in two. Since these precision tools are used in genetic engineering and genome editing, information about their mechanism of action at all stages of DNA recognition and phosphodiester bond hydrolysis is essential. For the first time, fast kinetics of the Nt.BspD6I interaction with DNA were studied by the stopped-flow technique, and changes of optical characteristics were registered for the enzyme or DNA molecules. The role of divalent metal cations was estimated at all steps of Nt.BspD6I–DNA complex formation. It was demonstrated that divalent metal ions are not required for the formation of a non-specific complex of the protein with DNA. Nt.BspD6I bound five-fold more efficiently to its recognition site in DNA than to a random DNA. DNA bending was confirmed during the specific binding of Nt.BspD6I to a substrate. The optimal size of Nt.BspD6I’s binding site in DNA as determined in this work should be taken into account in methods of detection of nucleic acid sequences and/or even various base modifications by means of NEs.

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Investigation of site-specific DNA binding with nicking endonuclease Nt.BspD6I at single molecule level by atomic force microscopy

Cited by 4 publications

References 9 publications

Modes of Escherichia coli Dps Interaction with DNA as Revealed by Atomic Force Microscopy

Modes of Escherichia coli Dps Interaction with DNA as Revealed by Atomic Force Microscopy

Peculiarities of the interaction of the restriction endonuclease BspD6I with DNA containing its recognition site

Kinetic Analysis of the Interaction of Nicking Endonuclease BspD6I with DNA

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