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Perevyazova, T. A.; Rogulin, E. A.; Zheleznaya, L. A.; Matvienko, N. I.

doi:10.1023/a:1026008528310

Cited by 15 publications

(5 citation statements)

References 5 publications

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“…Nt.BstSEI and isoschizomer Nt.BstNBI (GAGTCN 4 ↓) were found in two strains of B. stearothermophilus (21,22). Nt.BspD6I, an isoschizomer of Nt.BstNBI with identical amino acid sequence has also been cloned (23). Nt.BstNBI was thought to be a nicking enzyme that had lost the ability to dimerize because the enzyme purified from the native strain was a NEase.…”

Section: Discussionmentioning

confidence: 99%

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

Xu¹,

Zhu²,

Zhang³

et al. 2007

Nucleic Acids Research

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BsrDI and BtsI restriction endonucleases recognize and cleave double-strand DNA at the sequences GCAATG (2/0) and GCAGTG (2/0), respectively. We have purified and partially characterized these two enzymes, and analyzed the genes that encode them. BsrDI and BtsI are unusual in two respects: each cleaves DNA as a heterodimer of one large subunit (B subunit) and one small subunit (A subunit); and, in the absence of their small subunits, the large subunits behave as sequence-specific DNA nicking enzymes and only nick the bottom strand of the sequences at these respective positions: GCAATG (−/0) and GCAGTG (−/0). We refer to the single subunit, the bottom-strand nicking forms as ‘hemidimers’. Amino acid sequence comparisons reveal that BsrDI and BtsI belong to a family of restriction enzymes that possess two catalytic sites: a canonical PD-Xn-EXK and a second non-canonical PD-Xn-E-X12-QR. Interestingly, the other family members, which include BsrI (ACTGG 1/−1) and BsmI/Mva1269I (GAATGC 1/−1) are single polypeptide chains, i.e. monomers, rather than heterodimers. In BsrDI and BtsI, the two catalytic sites are found in two separate subunits. Site-directed mutagenesis confirmed that the canonical catalytic site located at the N-terminus of the large subunit is responsible for the bottom-strand cleavage, whereas the non-canonical catalytic site located in the small subunit is responsible for hydrolysis of the top strand. Top-strand specific nicking variants, Nt.BsrDI and Nt.BtsI, were successfully engineered by combining the catalytic-deficient B subunit with wild-type A subunit.

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Section: Discussionmentioning

confidence: 99%

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

Xu¹,

Zhu²,

Zhang³

et al. 2007

Nucleic Acids Research

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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).…”

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confidence: 99%

Nicking endonucleases

Zheleznaya

Качалова

Artyukh

et al. 2009

Biochemistry Moscow

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“…Complexes of Nt.BspD6I moved from cathode to anode due to the negative charge of the DNA duplex; thus, the samples were prepared without Coomassie G 250 in these experi ments. Calcium ions were added to the buffer to pre vent hydrolysis by Nt.BspD6I while maintaining bind ing specificity [10,11]. Complex formation was detected in 8% nondenaturing PAGE autoradiograph ically (Fig.…”

Section: Resultsmentioning

confidence: 92%

“…Nt.BspD6I has the same amino acid sequence as Nt.BstNBI [10]. It was shown that Nt.BstNBI was not able to form a dimer at low concentrations either in the free state or interacting with the specific DNA [11].…”

Section: Introductionmentioning

confidence: 99%

Oligomerization of site-specific nicking endonuclease BspD6I at high protein concentrations

et al. 2012

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Ability of site-specific nickase BspD6I (Nt.BspD6I) to oligomerize at concentrations > or = 0.5 microM (> or = 0.035 mg/mL) is studied. Three states of Nt.BspD6I are registered via electrophoretic studies both in the presence and in the absence of DNA. Estimation of their molecular mass allows assigning them as a monomer, a dimer and a trimer. Both dimeric and monomeric Nt.BspD6I are shown to hydrolyze its DNA substrate with the identical specificity. Calculation of the electrostatic potential distribution on the Nt.BspD6I globule surface shows that the protein molecule is a dipole. The Nt. BspD6I oligomeric forms are likely to be the result of ionic protein interactions.

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Cited by 15 publications

References 5 publications

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

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

Nicking endonucleases

Oligomerization of site-specific nicking endonuclease BspD6I at high protein concentrations

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