EcoRII Restriction Endonuclease Forms Specific Contacts to the Bases of Its Target Sequence Flipped from DNA in a Transition Complex with Photoactivatable Substrates

2021 ◽  
Vol 47 (2) ◽  
pp. 367-375
Author(s):  
O. V. Kirsanova ◽  
F. V. Subach ◽  
A. G. Loiko ◽  
R. I. Eritja ◽  
E. S. Gromova
Keyword(s):  
Restriction Endonuclease ◽  
Target Sequence ◽  
Transition Complex

scholarly journals In vitro analysis of site specific nuclease selectivity by NGS

2021 ◽  
Vol 8 (4) ◽  
pp. 235-242
Author(s):  
Vincent Brondani ◽  
Keyword(s):  
Restriction Endonuclease ◽  
Genome Engineering ◽  
Direct Interaction ◽  
Target Sequence ◽  
Vitro Assay ◽  
Guide Rna ◽  
Phosphate Backbone ◽  
Target Dna ◽  
Vitro Analysis

<abstract> <p>Nucleases currently used in genome engineering induce hydrolysis of DNA phosphate backbone in a sequence-specific manner. The RNA guided nucleases describe today are recognizing a sequence with two distinct molecular interactions: first, like a restriction endonuclease, by direct interaction between the protein and the DNA; and second, by hybridization of the guide RNA with the target DNA sequence. Here we report an in vitro assay to assess the cleavage specificity and the selectivity of the nucleases. The assay is designed using a plasmid encompassing the DNA target site degenerated at positions determined on structural feature. The results demonstrate that the Cpf1 RNA guided nuclease is highly specific for the target sequence, nevertheless its substrate selectivity is low compare to a restriction endonuclease.</p> </abstract>

scholarly journals Restriction endonuclease MvaI is a monomer that recognizes its target sequence asymmetrically

2007 ◽  
Vol 35 (6) ◽  
pp. 2035-2046 ◽  
Author(s):  
Magdalena Kaus-Drobek ◽  
Honorata Czapinska ◽  
Monika Sokołowska ◽  
Gintautas Tamulaitis ◽  
Roman H. Szczepanowski ◽  
...  
Keyword(s):  
Restriction Endonuclease ◽  
Target Sequence

Electron microscopic assays of restriction endonuclease cutting, exonuclease digestion, and hybridization

1984 ◽  
Vol 42 ◽  
pp. 686-687
Author(s):  
Mark Hannibal ◽  
Jacob Varkey ◽  
Michael Beer
Keyword(s):  
Electron Microscope ◽  
Low Temperature ◽  
Restriction Endonuclease ◽  
Double Helix ◽  
Test Material ◽  
Electron Microscopic ◽  
Exonuclease Iii ◽  
Dna Molecule ◽  
Linear Molecules ◽  
Exonuclease Digestion

Workman and Langmore have recently proposed a procedure for isolating particular chromatin fragments. The method requires restriction endonuclease cutting of the chromatin and a probe, their digestion with two exonucleases which leave complimentary single strand termini and low temperature hybridization of these. We here report simple electron microscopic monitoring of the four reactions involved.Our test material was ϕX-174 RF DNA which is cut once by restriction endonuclease Xho I. The conversion of circles to linear molecules was followed in Kleinschmidt spreads. Plate I shows a circular and a linear DNA molecule. The rate of cutting is shown in Figure 1.After completion of the endonuclease cutting, one portion of the DNA was treated with exonuclease III, an enzyme known to digest the 3' terminals of double helical DNA. Aliquots when examined in the electron microscope reveal a decreasing length of double helix and increasing bushes at the ends.

Use of Antibody to aid Visualization of Protein at the Termini of Bacteriophage Ø29 DNA

1980 ◽  
Vol 38 ◽  
pp. 540-541
Author(s):  
Dwight Anderson ◽  
Charlene Peterson ◽  
Gursaran Notani ◽  
Bernard Reilly
Keyword(s):  
Electron Microscopy ◽  
Bacillus Subtilis ◽  
Dna Synthesis ◽  
Restriction Endonuclease ◽  
Protein Complex ◽  
Protein Product ◽  
Viral Dna ◽  
Small Proteins ◽  
Antibody Labeling ◽  
Subtilis Bacteriophage

The protein product of cistron 3 of Bacillus subtilis bacteriophage Ø29 is essential for viral DNA synthesis and is covalently bound to the 5’-termini of the Ø29 DNA. When the DNA-protein complex is cleaved with a restriction endonuclease, the protein is bound to the two terminal fragments. The 28,000 dalton protein can be visualized by electron microscopy as a small dot and often is seen only when two ends are in apposition as in multimers or in glutaraldehyde-fixed aggregates. We sought to improve the visibility of these small proteins by use of antibody labeling.

ASSESSMENT OF EFFICIENCY AND OFF-TARGET ACTIVITY OF CRISPR/CAS RIBONUCLEOPROTEIN COMPLEXES

2020 ◽  
Author(s):  
Y.V. Mikhaylova ◽  
◽  
M.A. Tyumentseva ◽  
A.A. Shelenkov ◽  
Y.G. Yanushevich ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Correct Choice ◽  
Target Sequence ◽  
Dna Breaks ◽  
Gene Encoding ◽  
Guide Rna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Chemokine Receptor Ccr5 ◽  
Target Activity

In this study, we assessed the efficiency and off-target activity of the CRISPR/CAS complex with one of the selected guide RNAs using the CIRCLE-seq technology. The gene encoding the human chemokine receptor CCR5 was used as a target sequence for genome editing. The results of this experiment indicate the correct choice of the guide RNA and efficient work of the CRISPR- CAS ribonucleoprotein complex used. CIRCLE-seq technology has shown high sensitivity compared to bioinformatic methods for predicting off-target activity of CRISPR/CAS complexes. We plan to evaluate the efficiency and off-target activity of CRISPR/CAS ribonucleoprotein complexes with other guide RNAs by slightly adjusting the CIRCLE-seq-technology protocol in order to reduce nonspecific DNA breaks and increase the number of reliable reads.

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