Affinity Modification of the Restriction Endonuclease SsoII by 2′-Aldehyde-Containing Double Stranded DNAs

Sudina, Anna; Zatsepin, Timofei S.; Pingoud, Vera; Pingoud, Alfred; Oretskaya, Tatiana S.; Кубарева, Е. А.

doi:10.1007/s10541-005-0206-0

Cited by 5 publications

(3 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The conclusions about DNA binding drawn from the X‐ray structure analysis of R.Ecl18kI in complex with its substrate (PDB code 2fqz; Fig. ) are in full agreement with our biochemical data for R.SsoII DNA binding . Thus, the structural data obtained for R.Ecl18kI can be used to choose amino acids of R.SsoII for further modification.…”

Section: Resultssupporting

confidence: 83%

Thermo-switchable activity of the restriction endonuclease SsoII achieved by site-directed enzyme modification

et al. 2013

View full text Add to dashboard Cite

In this work, the possibility of constructing a thermoswitchable enzyme according to the "molecular gate" strategy is demonstrated. The approach is based on the covalent attachment of oligodeoxyribonucleotides to cysteine residues of an enzyme adjacent to its active center to form a temporal barrier for enzyme-substrate complex formation. The activity of the modified enzyme that had been studied here-the restriction endonuclease SsoII (R.SsoII)-was diminished by a factor of 180 at 25 that almost abolished the enzymatic activity when compared with the unmodified enzyme. However, heating of the modified enzyme to 45 resulted in a 30-fold increase of activity. The activity of unmodified R.SsoII also increased on heating from 25 to 45; however, the difference did not exceed a factor of 3-4. The changes in enzymatic activity observed were shown to be reversible for both the unmodified and the modified R.SsoII. Variation of the length and the sequence of the attached oligodeoxyribonucleotides might allow greater modulation of the activity of DNA-protein conjugates.

show abstract

Section: Resultssupporting

confidence: 83%

Thermo-switchable activity of the restriction endonuclease SsoII achieved by site-directed enzyme modification

et al. 2013

View full text Add to dashboard Cite

show abstract

“…We demonstrate that manipulation of in vitro conditions enables DpnI to bind but not cut DNA containing its target sequence. While the binding affinity of DpnI has not been determined, several restriction enzymes have been measured in the picomolar [27] , [28] to nanomolar range [29] , [30] and our results support the use of restriction enzymes as strong and specific DNA binding proteins.…”

Section: Discussionsupporting

confidence: 78%

Selective Microbial Genomic DNA Isolation Using Restriction Endonucleases

et al. 2014

View full text Add to dashboard Cite

To improve the metagenomic analysis of complex microbiomes, we have repurposed restriction endonucleases as methyl specific DNA binding proteins. As an example, we use DpnI immobilized on magnetic beads. The ten minute extraction technique allows specific binding of genomes containing the DpnI Gm6ATC motif common in the genomic DNA of many bacteria including γ-proteobacteria. Using synthetic genome mixtures, we demonstrate 80% recovery of Escherichia coli genomic DNA even when only femtogram quantities are spiked into 10 µg of human DNA background. Binding is very specific with less than 0.5% of human DNA bound. Next Generation Sequencing of input and enriched synthetic mixtures results in over 100-fold enrichment of target genomes relative to human and plant DNA. We also show comparable enrichment when sequencing complex microbiomes such as those from creek water and human saliva. The technique can be broadened to other restriction enzymes allowing for the selective enrichment of trace and unculturable organisms from complex microbiomes and the stratification of organisms according to restriction enzyme enrichment.

show abstract

“…GGTCTCGAGTCTTCT↓CAAGGT CCAGAGCTCAGAAGA-GTTCCA After the nicking reaction, the mixtures were separated by 20% PAGE with a plate size of 200 × 200 × 1 mm 3 (acrylamide:N,N -bisacrylamide at 19:1) in the presence of 7 M urea at a field force of 30 V/cm in TBE buffer [13]. Before loading onto the gel, the reaction mixtures were heated for 3 min at 95 • C. On a Typhoon FLA 9500 instrument (GE Healthcare, USA), the fluorescence of the zones containing DNA was detected and analysed using the ImageQuant software (GE Healthcare, UK) (https://www.cytivalifesciences.com/en/us/shop/protein-analysis/ molecular-imaging-for-proteins/imaging-software/imagequant-tl-8-2-image-analysis-softwarep-09518, accessed on 21 September 2021).…”

Section: Iii-21mentioning

confidence: 99%

Kinetic Analysis of the Interaction of Nicking Endonuclease BspD6I with DNA

et al. 2021

View full text Add to dashboard Cite

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.

show abstract

Affinity Modification of the Restriction Endonuclease SsoII by 2′-Aldehyde-Containing Double Stranded DNAs

Cited by 5 publications

References 17 publications

Thermo-switchable activity of the restriction endonuclease SsoII achieved by site-directed enzyme modification

Thermo-switchable activity of the restriction endonuclease SsoII achieved by site-directed enzyme modification

Selective Microbial Genomic DNA Isolation Using Restriction Endonucleases

Kinetic Analysis of the Interaction of Nicking Endonuclease BspD6I with DNA

Contact Info

Product

Resources

About