Discovery of natural nicking endonucleases Nb.BsrDI and Nb.BtsI and engineering of top-strand nicking variants from BsrDI and BtsI

Xu, Shuang-yong; Zhu, Zhenyu; Zhang, Penghua; Chan, Siu-Hong; Samuelson, James C.; Xiao, Jianping; Ingalls, Debra; Wilson, Geoffrey G.

doi:10.1093/nar/gkm481

Cited by 49 publications

(55 citation statements)

References 32 publications

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“…Detection of heterodimeric endonucleases BtsI and BsrDI [22] whose properties are similar to those of BspD6I, found by us, shows that heterodimeric endonu cleases, one subunit of which in the absence of the other exhibits activity of nicking endonuclease, are probably widespread in nature. However, it is rather difficult to reg ister the presence of heterodimeric endonucleases in bac terial cell lysates.…”

Section: Resultssupporting

confidence: 73%

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Nicking endonucleases

Zheleznaya

Качалова

Artyukh

et al. 2009

Biochemistry Moscow

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Section: Resultssupporting

confidence: 73%

“…Soon two other het erodimeric endonucleases, BsrDI and BtsI, were found [22], and one of their subunits exhibits nicking activity. BsrDI was isolated from B. stearothermophilus D70.…”

Section: Nicking Endonucleases -Subunits Of Heterodimeric Restrictionmentioning

confidence: 99%

Nicking endonucleases

Zheleznaya

Качалова

Artyukh

et al. 2009

Biochemistry Moscow

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“…The oligos are then amplified via PCR, which can be carried out with or without an emulsion (Materials and Methods) (36). Importantly, one of the primers contains a 5′-conjugated fluorophore, whereas the other contains the recognition site for a nicking endonuclease (NE) (37), which provides a strategy for making Oligopaints single stranded. As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…3D), whereas a 75,000-oligo subset of that pool has been combined with fluorescently labeled wheat germ agglutinin Each oligonucleotide in the library is composed of 32 bases of genomic sequence flanked by 21-base primer sequences. One of the primers carries a 5′ fluorophore, whereas the other contains a recognition site for a nicking endonuclease (NE) (37). A nicking reaction followed by denaturing gel electrophoresis yields 53-base ssDNAs.…”

Section: Oligopaints Robustly Label Interphase and Metaphase Chromosomentioning

confidence: 99%

Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes

Beliveau

Joyce

Apostolopoulos

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

398

472

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A host of observations demonstrating the relationship between nuclear architecture and processes such as gene expression have led to a number of new technologies for interrogating chromosome positioning. Whereas some of these technologies reconstruct intermolecular interactions, others have enhanced our ability to visualize chromosomes in situ. Here, we describe an oligonucleotide-and PCR-based strategy for fluorescence in situ hybridization (FISH) and a bioinformatic platform that enables this technology to be extended to any organism whose genome has been sequenced. The oligonucleotide probes are renewable, highly efficient, and able to robustly label chromosomes in cell culture, fixed tissues, and metaphase spreads. Our method gives researchers precise control over the sequences they target and allows for single and multicolor imaging of regions ranging from tens of kilobases to megabases with the same basic protocol. We anticipate this technology will lead to an enhanced ability to visualize interphase and metaphase chromosomes.T he role of chromosome positioning in gene regulation and chromosome stability is fueling a growing interest in technologies that reveal the in situ organization of the genome. Among these technologies are chromosome conformation capture (3C) (1) and its several iterations, such as Hi-C (2), which are applied to populations of nuclei to identify chromosomal regions that are in close proximity to each other (3, 4). Another technology is fluorescence in situ hybridization (FISH), wherein nucleic acids are targeted by fluorescently labeled probes and then visualized via microscopy; this technology is an extension of methods that once used radioactive probes and autoradiography but have since been adapted to use nonradioactive labels (5-11). FISH is a single-cell assay, making it especially powerful for the detection of rare events that might otherwise be lost in mixed or asynchronous populations of cells. In addition, because FISH is applied to fixed cells, it can reveal the positioning of chromosomes relative to nuclear, cytoplasmic, and even tissue structures. FISH can also be used to visualize RNA, permitting the simultaneous assessment of gene expression, chromosome position, and protein localization.FISH probes are typically derived from cloned genomic regions or flow-sorted chromosomes, which are labeled directly via nick translation or PCR in the presence of fluorophore-conjugated nucleotides or labeled indirectly with nucleotide-conjugated haptens, such as biotin and digoxigenin, and then visualized with secondary detection reagents. Probe DNA is often fragmented into ∼150-to 250-bp pieces to facilitate its penetration into fixed cells (12) and, as many genomic clones contain repetitive sequences that occur abundantly in the genome, hybridization is typically performed in the presence of unlabeled repetitive DNA (13). Another limitation to clone-based probes is that the genomic regions that can be visualized with them are restricted by the availability of clones and the size of ...

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“…Nickases Nb.BsrD1 and Nb.BtsI have been enginnered from their restriction enzymes, BsrD1 and BtsI (Xu ., 2007). Along similar lines, a nickase N.AlwI has been engineered from its restriction endonuclease, AlwI (Xu ., 2001).…”

Section: Isolation Of Strain and Plasmidsmentioning

confidence: 99%

Isolation of plasmid from a Gram negative bacteria Aeromonas dhakensis strain F2S2-1 and its partial characterization

Nadiga¹,

Vaidyanathan²,

Thangavelu³

2017

JEB

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Aim: Methodology:Results: Interpretation:A plasmid was isolated from strain F2S2-1 to understand their function in the host. The main objective of the study was to isolate and sequence the plasmid, predict genes and proteins encoded and to experimentally prove their biochemical function.The strain F2S2-1 plasmid was sequenced by Next Generation Sequencing (NGS) on Illumina Miseq platform. Bioinformatics analysis was carried out to predict the putative gene and encoded protein functions. The NspV like endonuclease was recombinantly expressed in , partially purified and characterized for activity on plasmid substrates.Plasmid sequencing and bioinformatics analysis predicted the presence of NspV like endonuclease.Recombinant expression, purification and characterization of NspV like endonuclease revealed the Nicking endonuclease activity of the protein with plasmid substrates. The NspV like endonuclease described in this study has DNA nicking activity. this study would help in understanding better survival of sp. in its ecosystem and the role of host plamids. Aeromonas dhakensis Aeromonas dhakensis Escherichia coli AeromonasJournal Home page : www.jeb.co.in « E-mail : editor@jeb.co.in Journal of Environmental Biology

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Discovery of natural nicking endonucleases Nb.BsrDI and Nb.BtsI and engineering of top-strand nicking variants from BsrDI and BtsI

Cited by 49 publications

References 32 publications

Nicking endonucleases

Nicking endonucleases

Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes

Isolation of plasmid from a Gram negative bacteria Aeromonas dhakensis strain F2S2-1 and its partial characterization

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