Primary Structure of NM.BstSEI Operon from Bacillus stearothermophilus, the Producer of N.BstSEI Site-Specific Nicking Endonuclease

Gololobova, N. S.; Okhapkina, S. S.; Abdurashitov, M. A.; Degtyarev, S. Kh.

doi:10.1007/s11008-005-0103-z

Cited by 6 publications

(3 citation statements)

References 9 publications

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“…These MTases gene sequences are completely identical with the M1.BstSEI and M2.BstSEI genes found in Bacillus stearothermophilus (T.A. Perevyazova, unpublished data, [34]). Chernukhin et al [35] showed, that purified recombinant M2.BstSEI alone (in the absence of M1.BstSEI) is capable to modify adenine in both DNA strands of double-stranded 5′-GASTC-3′ (S = G or C) sequence.…”

Section: 22supporting

confidence: 58%

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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Section: 22supporting

confidence: 58%

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 high frequency of such combination of sites, in turn, is explained by the fact that the GAGTC sequence is incor porated in promoter sequences of T7 DNA. The second amazing fact is that sequencing of nickase N.BstNBI [6], N.BspD6I [7], and N.BstSEI [8] genes and adjacent regions has shown 100% coincidence between all these sequences. Such a coincidence can be due to the fact that all researchers were dealing with the same widespread strain (the enzymes were isolated in laboratories geo graphically remote from each other).…”

mentioning

confidence: 99%

Nicking endonucleases

Zheleznaya

Качалова

Artyukh

et al. 2009

Biochemistry Moscow

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“…The first two natural NEases, Nt.CviPII and Nt.CviQII, were found from infected cells of Chlorella viruses [2, 3]. Other natural NEases are Nt.BstNBI/Nt.BspD6I/N.BstSEI, Nb.BsrDI, and Nb.BtsI, which are the large subunits of their respective REases [4-7] (G.Wilson, unpublished results). NEases have also been engineered from heterodimeric REases BbvCI and Bpu10I by inactivation of the top-strand or bottom-strand catalytic site [8] (Janulaitis A et al (2005) Strand-specific polynucleotide nickases, US patent number 6,867,028).…”

Section: Introductionmentioning

confidence: 99%

Engineering BspQI nicking enzymes and application of N.BspQI in DNA labeling and production of single-strand DNA

Zhang

Too

Samuelson

et al. 2010

Protein Expression and Purification

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BspQI is a thermostable Type IIS restriction endonuclease (REase) with the recognition sequence 5' GCTCTTC N1/N4 3'. Here we report the cloning and expression of the bspQIR gene for the BspQI restriction enzyme in E. coli. Alanine scanning of the BspQI charged residues identified a number of DNA nicking variants. After sampling combinations of different amino acid substitutions, an Nt.BspQI triple mutant (E172A/E248A/E255K) was constructed with predominantly top-strand DNA nicking activity. Furthermore, a triple mutant of BspQI (Nb.BspQI, N235A/K331A/R428A) was engineered to create a bottom-strand nicking enzyme. In addition, we demonstrated the application of Nt.BspQI in optical mapping of single DNA molecules. Nt or Nb.BspQI-nicked dsDNA can be further digested by E. coli exonuclease III to create ssDNA for downstream applications. BspQI contains two potential catalytic sites: a top-strand catalytic site (Ct) with a D-H-N-K motif found in the HNH endonuclease family and a bottom-strand catalytic site (Cb) with three scattered Glu residues. BlastP analysis of proteins in Genbank indicated a putative restriction enzyme with significant amino acid sequence identity to BspQI from the sequenced bacterial genome Croceibacter atlanticus HTCC2559. This restriction gene was amplified by PCR and cloned into a T7 expression vector. Restriction mapping and run-off DNA sequencing of digested products from the partially purified enzyme indicated that it is an EarI isoschizomer with 6-bp recognition, which we named CatHI (CTCTTC N1/N4). Keywords alanine scanning; nicking endonuclease; CatHI; DNA labeling; optical mapping of DNA *Corresponding author Phone: 978-380-7287, Fax: 978-921-1350, xus@neb.com. Individual contributions: P. Zhang expressed wt BspQI and engineered Nt.BspQI and Nb.BspQI; P. Too purified Nb.BspQI, performed nicking assays and produced circular ssDNA by exonuclease III; Siu-Hong Chan cloned and purified CatHI; J. Samuelson constructed a BspQI genomic DNA libraries and performed the methylase selection procedure and "endo-blue" screening; T. Vincze analyzed the 454 sequence contigs; S. Doucette purified wt BspQI; S.Bäckström independently tested Nb.BspQI/exonuclease III on different plasmid constructs.

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Primary Structure of NM.BstSEI Operon from Bacillus stearothermophilus, the Producer of N.BstSEI Site-Specific Nicking Endonuclease

Cited by 6 publications

References 9 publications

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

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

Nicking endonucleases

Engineering BspQI nicking enzymes and application of N.BspQI in DNA labeling and production of single-strand DNA

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