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

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 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.

scholarly journals Characterization of LlaKI, a New Metal Ion-Independent Restriction Endonuclease from Lactococcus lactis KLDS4

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Abdelkarim Belkebir ◽  
Houssine Azeddoug
Keyword(s):  
Restriction Endonuclease ◽  
Lactococcus Lactis ◽  
Dna Cleavage ◽  
Metal Ion ◽  
Divalent Cations ◽  
Restriction Enzymes ◽  
Restriction Endonucleases ◽  
Size Exclusion ◽  
Type Ii ◽  
Divalent Metal Ions

Requirement of divalent cations for DNA cleavage is a general feature of type II restriction enzymes with the exception of few members of this group. A new type II restriction endonuclease has been partially purified from Lactococcus lactis KLDS4. The enzyme was denoted as LlaKI and showed to recognize and cleave the same site as FokI. The enzyme displayed a denatured molecular weight of 50 kDa and behaved as a dimer in solution as evidenced by the size exclusion chromatography. To investigate the role of divalent cations in DNA cleavage by LlaKI, digestion reactions were carried out at different Mg2+, Mn2+, and Ca2+ concentrations. Unlike most of type II restriction endonucleases, LlaKI did not require divalent metal ions to cleave DNA and is one of the few metal-independent restriction endonucleases found in bacteria. The enzyme showed near-maximal levels of activity in 10 mM Tris-HCl pH 7.9, 50 mM NaCl, 10 mM MgCl2, and 1 mM dithiothreitol at 30°C. The presence of DNA modification was also determined and was correlated with the correspondent restriction enzyme.

scholarly journals How cyanophage S-2L rejects adenine and incorporates 2-aminoadenine to saturate hydrogen bonding in its DNA

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Dariusz Czernecki ◽  
Pierre Legrand ◽  
Mustafa Tekpinar ◽  
Sandrine Rosario ◽  
Pierre-Alexandre Kaminski ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Hydrogen Bonding ◽  
Crystal Structures ◽  
Active Site ◽  
Metal Ion ◽  
Restriction Endonucleases ◽  
Structural Element

AbstractBacteriophages have long been known to use modified bases in their DNA to prevent cleavage by the host’s restriction endonucleases. Among them, cyanophage S-2L is unique because its genome has all its adenines (A) systematically replaced by 2-aminoadenines (Z). Here, we identify a member of the PrimPol family as the sole possible polymerase of S-2L and we find it can incorporate both A and Z in front of a T. Its crystal structure at 1.5 Å resolution confirms that there is no structural element in the active site that could lead to the rejection of A in front of T. To resolve this contradiction, we show that a nearby gene is a triphosphohydolase specific of dATP (DatZ), that leaves intact all other dNTPs, including dZTP. This explains the absence of A in S-2L genome. Crystal structures of DatZ with various ligands, including one at sub-angstrom resolution, allow to describe its mechanism as a typical two-metal-ion mechanism and to set the stage for its engineering.

scholarly journals Analysis of the HindIII-catalyzed reaction by time-resolved crystallography

2015 ◽  
Vol 71 (2) ◽  
pp. 256-265 ◽  
Author(s):  
Takashi Kawamura ◽  
Tomoki Kobayashi ◽  
Nobuhisa Watanabe
Keyword(s):  
Electron Density ◽  
Active Site ◽  
Dna Cleavage ◽  
Metal Ion ◽  
Manganese Ions ◽  
Metal Ion Binding ◽  
Time Resolved ◽  
Manganese Ion ◽  
Ion Binding Sites ◽  
Metal Ion Binding Sites

In order to investigate the mechanism of the reaction catalyzed by HindIII, structures of HindIII–DNA complexes with varying durations of soaking time in cryoprotectant buffer containing manganese ions were determined by the freeze-trap method. In the crystal structures of the complexes obtained after soaking for a longer duration, two manganese ions, indicated by relatively higher electron density, are clearly observed at the two metal ion-binding sites in the active site of HindIII. The increase in the electron density of the two metal-ion peaks followed distinct pathways with increasing soaking times, suggesting variation in the binding rate constant for the two metal sites. DNA cleavage is observed when the second manganese ion appears, suggesting that HindIII uses the two-metal-ion mechanism, or alternatively that its reactivity is enhanced by the binding of the second metal ion. In addition, conformational change in a loop near the active site accompanies the catalytic reaction.

scholarly journals Fluorophore ATCUN complexes: combining agent and probe for oxidative DNA cleavage

2015 ◽  
Vol 51 (62) ◽  
pp. 12395-12398 ◽  
Author(s):  
C. Wende ◽  
N. Kulak
Keyword(s):  
Dna Cleavage ◽  
Metal Ion ◽  
Fluorescent Reporter ◽  
Dna Cleaving ◽  
Oxidative Dna Cleavage

A Cu(ii)-based peptidic DNA cleaving agent equipped with a Cu(ii)-sensing fluorescent reporter allows monitoring the fate of the nucleolytic metal ion.

A New Method for Determining the Stereochemistry of DNA Cleavage Reactions:  Application to theSfiI andHpaII Restriction Endonucleases and to the MuA Transposase†

Biochemistry ◽  
1999 ◽  
Vol 38 (14) ◽  
pp. 4640-4648 ◽  
Author(s):  
Kiyoshi Mizuuchi ◽  
Timothy J. Nobbs ◽  
Stephen E. Halford ◽  
Kenji Adzuma ◽  
Jun Qin
Keyword(s):  
Dna Cleavage ◽  
Restriction Endonucleases ◽  
New Method ◽  
Cleavage Reactions

Evidence for substrate-assisted catalysis in the DNA cleavage of several restriction endonucleases

Gene ◽  
1995 ◽  
Vol 157 (1-2) ◽  
pp. 157-162 ◽  
Author(s):  
Albert Jeltsch ◽  
Milda Pleckaityte ◽  
Ursel Selent ◽  
Heiner Wolfes ◽  
Virginius Siksnys ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Restriction Endonucleases
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