scholarly journals Restriction endonucleases that cleave RNA/DNA heteroduplexes bind dsDNA in A-like conformation

2020 ◽  
Vol 48 (12) ◽  
pp. 6954-6969
Author(s):  
Marlena Kisiala ◽  
Monika Kowalska ◽  
Michal Pastor ◽  
Henryk J Korza ◽  
Honorata Czapinska ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Metal Ion ◽  
Restriction Endonucleases ◽  
Dna Duplexes ◽  
Systematic Screening ◽  
Oh Groups ◽  
Dna Cleavage Activity ◽  
Target Dna ◽  
Specific Complex ◽  
Catalytic Metal

Abstract Restriction endonucleases naturally target DNA duplexes. Systematic screening has identified a small minority of these enzymes that can also cleave RNA/DNA heteroduplexes and that may therefore be useful as tools for RNA biochemistry. We have chosen AvaII (G↓GWCC, where W stands for A or T) as a representative of this group of restriction endonucleases for detailed characterization. Here, we report crystal structures of AvaII alone, in specific complex with partially cleaved dsDNA, and in scanning complex with an RNA/DNA hybrid. The specific complex reveals a novel form of semi-specific dsDNA readout by a hexa-coordinated metal cation, most likely Ca2+ or Mg2+. Substitutions of residues anchoring this non-catalytic metal ion severely impair DNA binding and cleavage. The dsDNA in the AvaII complex is in the A-like form. This creates space for 2′-OH groups to be accommodated without intra-nucleic acid steric conflicts. PD-(D/E)XK restriction endonucleases of known structure that bind their dsDNA targets in the A-like form cluster into structurally similar groups. Most such enzymes, including some not previously studied in this respect, cleave RNA/DNA heteroduplexes. We conclude that A-form dsDNA binding is a good predictor for RNA/DNA cleavage activity.

On the Divalent Metal Ion Dependence of DNA Cleavage by Restriction Endonucleases of the EcoRI Family

2009 ◽  
Vol 393 (1) ◽  
pp. 140-160 ◽  
Author(s):  
Vera Pingoud ◽  
Wolfgang Wende ◽  
Peter Friedhoff ◽  
Monika Reuter ◽  
Jürgen Alves ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Metal Ion ◽  
Divalent Metal ◽  
Restriction Endonucleases ◽  
Divalent Metal Ion

scholarly journals Synthesis, Characterization, Antimicrobial, DNA Cleavage and Fluorescent Activity of Metal ion(II) Coordinate with 2H-Chromene Azo novel ligand

2020 ◽  
Vol 11 (2) ◽  
pp. 1953-1960
Author(s):  
Huda Muayad Nafea ◽  
Ali Muayad Nafea Al- Kawaz
Keyword(s):  
Dna Cleavage ◽  
Metal Ion ◽  
Condensation Reaction ◽  
Free Ligand ◽  
Cleavage Activity ◽  
Dna Cleavage Activity ◽  
Uv Visible ◽  
Group 2 ◽  
Conductance Measuring ◽  
Cobalt And Nickel

A new ligand 2H-chromene containing azo group 2-(4-nitrophenyl)-N-(4-(phenyldiazenyl)-2H-chromen-4-amine (AH), were synthesized from the condensation reaction (1:2)of 2'-hydroxychalcone and p-aminoazobenzene. Co(II), Cu(II) and Ni(II) complexes of the new ligand have been synthesized and characterized using C.H.N. analysis,1HNMR spectra, FT.IR, UV/Visible, magnetic susceptible, conductance measuring, and fluorescence spectral spectroscopy; 13CNMR spectroscopy of the ligand was also studied. Spectroscopic results revealed the 2H-Chromene Azo (AH) ligand behaves as monodentate chelating via the nitrogen atom of amine groupat position 4 having 1:1 [M:L] ratio; suggested that the cobalt and nickel complexes have the tetrahedralstructure and a distorted tetrahedralgeometry for the copper complex, indicating their non-electrolyte nature. The new ligand shows a fluorescence emissioncomparingwith this fluorescence quenching was noticed in its metal complexes. The antibacterial potency of the free ligand and its chelates with metal ion(II) were screened against E.coli, K.pneumoniae, Staph.aureus andB.Subtilis;The DNA cleavage activity of the free ligand and its 2H-chromene azo metal (II) complexes was performedby the gel electrophoresis process, which giventhat thesecompounds are effectiveupon DNA cleavage .

scholarly journals Metal ion dependence of DNA cleavage by SepMI and EhoI restriction endonucleases

2013 ◽  
Vol 168 (2) ◽  
pp. 99-105 ◽  
Author(s):  
Abdelkarim Belkebir ◽  
Houssine Azeddoug
Keyword(s):  
Dna Cleavage ◽  
Metal Ion ◽  
Restriction Endonucleases

Synthesis, characterization, antimicrobial, pesticidal and DNA cleavage activity of germanium(IV) derivatives of 3-(2-methyl-2,3-dihydro-benzthiazo-2-yl)-chromen-2-one and N′-[1-2-oxo-2H-chrome-3yl-ethylidene]-hydrazinecarbodithionic acid benzyl ester ligands

2011 ◽  
Vol 34 (5-6) ◽  
pp. 139-146 ◽  
Author(s):  
Latika Dawara ◽  
Nighat Fahmi ◽  
R.V. Singh
Keyword(s):  
Antimicrobial Activity ◽  
Metal Complexes ◽  
Dna Cleavage ◽  
Metal Ion ◽  
Benzyl Ester ◽  
Germanium Atom ◽  
Conventional Heating ◽  
Molar Ratio ◽  
Cleavage Activity ◽  
Dna Cleavage Activity

Abstract The new ligands 3-(2-methyl-2,3-dihydro-benzthiazo-2-yl)-chromen-2one (AcBzH) and N′-[1-2-oxo-2H-chrome-3yl-ethylidene]-hydrazinecarbodithionic acid benzyl ester (AcBDTZH) were prepared by the reaction of 3-acetyl-2H-chromen-2-one with 2-aminothiophenol and S-benzyl dithiocarbazate, respectively. The germanium(IV) complexes have been prepared by reacting Ph3GeCl and Me3GeCl in 1:1 molar ratio with these monofunctional bidentate AcBzH and AcBDTZH ligands by using microwave as well as conventional heating methods for comparison purposes. All the synthesized compounds were characterized by elemental analyses, melting point, IR, 1H-NMR 13C-NMR, mass and X-ray powder diffraction techniques. These studies showed that the ligands coordinated to the germanium atom in a monobasic bidentate manner and trigonal bipyramidal environment around the germanium atom have been established for the complexes. To evaluate the effect of metal ion upon chelation, both ligands and their complexes have been screened for their antimicrobial activity against the various pathogenic bacterial and fungal strains. The metal complexes have shown antimicrobial activity as compared to the free ligands. The pesticidal activity and DNA cleavage activity of both ligands and their metal complexes have been tested and discussed.

scholarly journals Active-Site Models of Streptococcus pyogenes Cas9 in DNA Cleavage State

2021 ◽  
Vol 8 ◽  
Author(s):  
Honghai Tang ◽  
Hui Yuan ◽  
Wenhao Du ◽  
Gan Li ◽  
Dongmei Xue ◽  
...  
Keyword(s):  
Active Site ◽  
Dna Cleavage ◽  
Metal Ion ◽  
Active Sites ◽  
Structural Similarity ◽  
Atomic Model ◽  
Nucleophilic Attack ◽  
Target Dna ◽  
Coordinated Water ◽  
Dna Complex

CRISPR-Cas9 is a powerful tool for target genome editing in living cells. Significant advances have been made to understand how this system cleaves target DNA. HNH is a nuclease domain, which shares structural similarity with the HNH endonuclease characterzied by a beta-beta-alpha-metal fold. Therefore, based on one- and two-metal-ion mechanisms, homology modeling and molecular dynamics (MD) simulation are suitable tools for building an atomic model of Cas9 in the DNA cleavage state. Here, by modeling and MD, we presented an atomic model of SpCas9–sgRNA–DNA complex with the cleavage state. This model shows that the HNH and RuvC conformations resemble their DNA cleavage state where the active-sites in the complex coordinate with DNA, Mg2+ ions, and water. Among them, residues D10, E762, H983, and D986 locate at the first shell of the RuvC active-site and interact with the ions directly, residues H982 or/and H985 are general (Lewis) bases, and the coordinated water is located at the positions for nucleophilic attack of the scissile phosphate. Meanwhile, this catalytic model led us to engineer a new SpCas9 variant (SpCas9-H982A + H983D) with reduced off-target effects. Thus, our study provided new mechanistic insights into the CRISPR-Cas9 system in the DNA cleavage state and offered useful guidance for engineering new CRISPR-Cas9 editing systems with improved specificity.

scholarly journals SYNTHESIS, CHARACTERISATION, THERMAL ANALYSIS AND DNA CLEAVAGE ACTIVITY OF A NOVELZINC (II) COMPLEX OF PYRAZOLE SCHIFF BASES

2018 ◽  
Vol 5 (2) ◽  
pp. 19-23
Author(s):  
Jayanthi Eswaran
Keyword(s):  
Schiff Base ◽  
Schiff Base Ligand ◽  
Dna Cleavage ◽  
Metal Ion ◽  
Acid Hydrazide ◽  
Single Step ◽  
Cleavage Activity ◽  
Dna Cleavage Activity ◽  
Shiff Base ◽  
Ft Ir

A hydrazone Schiff base Zn(II) metal complex is synthesised from the Shiff base ligand Thiophene-2-carboxylic acid hydrazide and 1, 3-diphenyl-1H-pyrazole-4-carboxaldehyde reacted together in 1:1 mole ratio to obtain Schiff base ligand (HL) which was subsequently, allowed to react with Zn(CH3COO)2.2H2O. The Schiff base ligand and its Zn (II) complex prepared were characterized on the basis of elemental nalysis,thermogravimetry, UV-Visible spectroscopy, FT-IR spectroscopy and NMR spectroscopy. IR spectrum of the zinc complex shows that the ligand (HL) is coordinated to the metal ion in monoanionicbidentate fashion with the 1:2 metal to ligand stoichiometry. The thermal behaviour of thecomplex shows a single step decomposition pattern leaving the respective ZnO residue. The DNA cleavage activity of the complex ismonitored using agarose gel lectrophoresis method which indicates the potential of the complex to cleave supercoiled DNA.

scholarly journals Structural and mechanistic insight into CRISPR-Cas9 inhibition by anti-CRISPR protein AcrIIC4

2021 ◽  
Author(s):  
Sungwon Hwang ◽  
Chuxi Pan ◽  
Bianca Garcia ◽  
Alan R Davidson ◽  
Trevor F Moraes ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Dna Cleavage ◽  
Three Dimensional ◽  
Cleavage Activity ◽  
Mechanistic Insight ◽  
Haemophilus Parainfluenzae ◽  
Dna Cleavage Activity ◽  
Target Dna ◽  
Dimensional Crystal ◽  
Insight Into

Phages, plasmids, and other mobile genetic elements express inhibitors of CRISPR-Cas immune systems, known as anti-CRISPR proteins, to protect themselves from targeted destruction. These anti-CRISPRs have been shown to function through very diverse mechanisms. In this work we investigate the activity of an anti-CRISPR isolated from a prophage in Haemophilus parainfluenzae that blocks CRISPR-Cas9 DNA cleavage activity. We determine the three-dimensional crystal struture of AcrIIC4 and show that it binds to the Cas9 Recognition Domain. This binding does not prevent the Cas9-anti-CRISPR complex from interacting with target DNA but does inhibit DNA cleavage. AcrIIC4 likely acts by blocking the conformational changes that allow the HNH and RuvC endonuclease domains to contact the DNA sites to be nicked.

scholarly journals Syntheses, Characterization, Antimicrobial and DNA Cleavage Activity of Cr(III) Complexes with Coumarin based Schiff Base Ligands

2021 ◽  
Vol 33 (12) ◽  
pp. 2983-2988
Author(s):  
Ramhari Meena ◽  
Anita Kumari ◽  
Naveen Sharma ◽  
Nighat Fahmi
Keyword(s):  
Schiff Base ◽  
Dna Cleavage ◽  
Metal Ion ◽  
Biological Activities ◽  
Spectroscopic Techniques ◽  
Molecular Weight Determination ◽  
Central Metal ◽  
Schiff Base Ligands ◽  
Cleavage Activity ◽  
Dna Cleavage Activity

A series of biologically important complexes of chromium(III) have been synthesized by the reaction of 3-formyl-4-chlorocoumarin hydrazinecarbothioamide (L1H) and 3-formyl-4-chlorocoumarin hydrazinecarboxamide (L2H) with CrCl3·6H2O in 1:1 and 1:2 molar ratio. All the complexes have been characterized by elemental analysis, molecular weight determination, melting point, conductivity measurements, electronic, IR, 1H NMR, 13C NMR and EPR spectroscopic techniques and X-ray diffraction. In vitro biological screening effects of the compounds were tested against the pathogenic bacterial and fungal species. Further, free ligands and their metal complexes have been screened for their DNA cleavage activity. A comparative study of the biological activities of the Schiff base ligands and their Cr(III) complexes indicates that the complexes exhibit higher antimicrobial and DNA cleavage activity than the free ligands. Physico-chemical studies and spectral data suggested a hexa-coordinated environment around the central metal ion.

scholarly journals Indirect Readout of DNA Controls Filamentation and Activation of a Sequence-Specific Endonuclease

2019 ◽  
Author(s):  
Smarajit Polley ◽  
Dmitry Lyumkis ◽  
N. C. Horton
Keyword(s):  
Conformational Changes ◽  
Dna Cleavage ◽  
Metal Ion ◽  
Structural Changes ◽  
Enzyme Mechanism ◽  
Sequence Specificity ◽  
Dna Sequence Specificity ◽  
Cleavage Activity ◽  
Dna Cleavage Activity ◽  
Indirect Readout

ABSTRACTFilament or run-on oligomer formation by enzymes is increasingly recognized as an important phenomenon with potentially unique regulatory properties and biological roles. SgrAI is an allosterically regulated type II restriction endonuclease that forms run-on oligomeric (ROO) filaments with enhanced DNA cleavage activity and altered sequence specificity. Here, we present the 3.5 Å cryo-electron microscopy structure of the ROO filament of SgrAI bound to a mimic of cleaved primary site DNA and Mg2+. Large conformational changes stabilize a second metal ion cofactor binding site within the catalytic pocket and facilitate assembling a higher-order enzyme form that is competent for rapid DNA cleavage. The structural changes illuminate the mechanistic origin of hyper-accelerated DNA cleavage activity within the filamentous SgrAI form. An analysis of the protein-DNA interface and the stacking of individual nucleotides reveals how indirect DNA readout within filamentous SgrAI enables recognition of substantially more nucleotide sequences than its low-activity form, thereby expanding DNA sequence specificity. Together, substrate DNA binding, indirect readout, and filamentation simultaneously enhance SgrAI’s catalytic activity and modulate substrate preference. This unusual enzyme mechanism may have evolved to perform the specialized functions of bacterial innate immunity in rapid defense against invading phage DNA without causing damage to the host DNA.

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