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.

Properties of the mammalian and yeast metal-ion transporters DCT1 and Smf1p expressed in Xenopus laevis oocytes

2001 ◽  
Vol 204 (6) ◽  
pp. 1053-1061 ◽  
Author(s):  
A. Sacher ◽  
A. Cohen ◽  
N. Nelson
Keyword(s):  
Xenopus Laevis ◽  
Metal Ions ◽  
Metal Ion ◽  
Divalent Cations ◽  
Ion Uptake ◽  
Xenopus Laevis Oocytes ◽  
Ion Transporters ◽  
Divalent Metal Ions ◽  
Transport Pathway ◽  
Metal Ion Uptake

Transition metals are essential for many metabolic processes, and their homeostasis is crucial for life. Metal-ion transporters play a major role in maintaining the correct concentrations of the various metal ions in living cells. Little is known about the transport mechanism of metal ions by eukaryotic cells. Some insight has been gained from studies of the mammalian transporter DCT1 and the yeast transporter Smf1p by following the uptake of various metal ions and from electrophysiological experiments using Xenopus laevis oocytes injected with RNA copies (c-RNA) of the genes for these transporters. Both transporters catalyze the proton-dependent uptake of divalent cations accompanied by a ‘slippage’ phenomenon of different monovalent cations unique to each transporter. Here, we further characterize the transport activity of DCT1 and Smf1p, their substrate specificity and their transport properties. We observed that Zn(2+) is not transported through the membrane of Xenopus laevis oocytes by either transporter, even though it inhibits the transport of the other metal ions and enables protons to ‘slip’ through the DCT1 transporter. A special construct (Smf1p-s) was made to enhance Smf1p activity in oocytes to enable electrophysiological studies of Smf1p-s-expressing cells. 54Mn(2+) uptake by Smf1p-s was measured at various holding potentials. In the absence of Na(+) and at pH 5.5, metal-ion uptake was not affected by changes in negative holding potentials. Elevating the pH of the medium to 6.5 caused metal-ion uptake to be influenced by the holding potential: ion uptake increased when the potential was lowered. Na(+) inhibited metal-ion uptake in accordance with the elevation of the holding potential. A novel clutch mechanism of ion slippage that operates via continuously variable stoichiometry between the driving-force pathway (H(+)) and the transport pathway (divalent metal ions) is proposed. The possible physiological advantages of proton slippage through DCT1 and of Na(+) slippage through Smf1p are discussed.

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 Use of Divalent Metal Ions in the DNA Cleavage Reaction of Human Type II Topoisomerases†

Biochemistry ◽  
2009 ◽  
Vol 48 (9) ◽  
pp. 1862-1869 ◽  
Author(s):  
Joseph E. Deweese ◽  
Amber M. Burch ◽  
Alex B. Burgin ◽  
Neil Osheroff
Keyword(s):  
Metal Ions ◽  
Dna Cleavage ◽  
Divalent Metal ◽  
Type Ii ◽  
Divalent Metal Ions ◽  
Cleavage Reaction ◽  
Human Type

DNA cleavage by the EcoRV restriction endonuclease: roles of divalent metal ions in specificity and catalysis

1999 ◽  
Vol 288 (1) ◽  
pp. 87-103 ◽  
Author(s):  
Geoffrey S. Baldwin ◽  
Richard B. Sessions ◽  
Symon G. Erskine ◽  
Stephen E. Halford
Keyword(s):  
Metal Ions ◽  
Restriction Endonuclease ◽  
Dna Cleavage ◽  
Divalent Metal ◽  
Divalent Metal Ions

scholarly journals A novel restriction endonuclease UnbI, a neoschizomer of Sau96I from an unidentified psychrofilic bacterium from Antarctica is inhibited by phosphate ions.

1997 ◽  
Vol 44 (4) ◽  
pp. 849-852 ◽  
Author(s):  
M Kawalec ◽  
P Borsuk ◽  
S Piechula ◽  
P P Stepień
Keyword(s):  
Low Temperature ◽  
Restriction Endonuclease ◽  
Bacterial Strain ◽  
Inorganic Phosphate ◽  
Restriction Enzymes ◽  
Type Ii ◽  
Temperature Optimum ◽  
Phosphate Ions ◽  
Sticky Ends

A novel type II restriction endonuclease UnbI was isolated from an unidentified psychrofilic bacterial strain from Antarctica. UnbI recognizes and cleaves the sequence 5'-GGNCC-3', producing 5 nucleotide long sticky ends. In this respect it differs from its neoschizomer Sau96I and all other restriction enzymes recognizing this sequence. UnbI has a relatively low temperature optimum of 15 degrees C to 20 degrees C and its activity is completely inhibited by inorganic phosphate.

The Novel Two-Metal-Ion Mechanism for Type II Topoisomerases by the QM/MM and Free Energy Study

2019 ◽  
Author(s):  
Jingheng Wu ◽  
Hui Chao ◽  
Yong Shen
Keyword(s):  
Free Energy ◽  
Metal Ion ◽  
Divalent Metal ◽  
Free Energy Calculations ◽  
Nucleophilic Attack ◽  
Type Ii ◽  
Divalent Metal Ions ◽  
Dna Scission ◽  
Dna Strand ◽  
High Level

Catalysis of Type II topoisomerase employs a combination of nucleobase and divalent metal ions with a long discussing two-metal-ion mechanism. High-level quantum mechanics/molecular mechanics (QM/MM) and thermodynamics cycle perturbation (QTCP) free energy calculations support an associative novel two-metal-ion mechanism and elucidate the catalytic roles of metal ion, in which one divalent metal ion stabilizes the phosphoric pentacovalent transition state and the 3’‒OH leaving group while the secondary one facilitates to reorganize the nearby hydrogen network and residues. The DNA scission is fast and exothermic that a stepwise pathway proceeds for the nucleophilic attack by Y805 following by the protonation of the ribose alkoxide, inducing the formation of a bending DNA strand. These findings advance the fundamental knowledge on topoisomerases and the development of targeting anticancer drugs.

The Novel Two-Metal-Ion Mechanism for Type II Topoisomerases by the QM/MM and Free Energy Study

2019 ◽  
Author(s):  
Jingheng Wu ◽  
Hui Chao ◽  
Yong Shen
Keyword(s):  
Free Energy ◽  
Metal Ion ◽  
Divalent Metal ◽  
Free Energy Calculations ◽  
Nucleophilic Attack ◽  
Type Ii ◽  
Divalent Metal Ions ◽  
Dna Scission ◽  
Dna Strand ◽  
High Level

Catalysis of Type II topoisomerase employs a combination of nucleobase and divalent metal ions with a long discussing two-metal-ion mechanism. High-level quantum mechanics/molecular mechanics (QM/MM) and thermodynamics cycle perturbation (QTCP) free energy calculations support an associative novel two-metal-ion mechanism and elucidate the catalytic roles of metal ion, in which one divalent metal ion stabilizes the phosphoric pentacovalent transition state and the 3’‒OH leaving group while the secondary one facilitates to reorganize the nearby hydrogen network and residues. The DNA scission is fast and exothermic that a stepwise pathway proceeds for the nucleophilic attack by Y805 following by the protonation of the ribose alkoxide, inducing the formation of a bending DNA strand. These findings advance the fundamental knowledge on topoisomerases and the development of targeting anticancer drugs.

scholarly journals Generation of DNA cleavage specificities of type II restriction endonucleases by reassortment of target recognition domains

2007 ◽  
Vol 104 (25) ◽  
pp. 10358-10363 ◽  
Author(s):  
S. Jurenaite-Urbanaviciene ◽  
J. Serksnaite ◽  
E. Kriukiene ◽  
J. Giedriene ◽  
C. Venclovas ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Target Recognition ◽  
Restriction Endonucleases ◽  
Type Ii

scholarly journals TRPM7 Provides an Ion Channel Mechanism for Cellular Entry of Trace Metal Ions

2002 ◽  
Vol 121 (1) ◽  
pp. 49-60 ◽  
Author(s):  
Mahealani K. Monteilh-Zoller ◽  
Meredith C. Hermosura ◽  
Monica J.S. Nadler ◽  
Andrew M. Scharenberg ◽  
Reinhold Penner ◽  
...  
Keyword(s):  
Trace Metal ◽  
Ion Channel ◽  
Metal Ions ◽  
Metal Ion ◽  
Divalent Cations ◽  
Divalent Metal Ions ◽  
Hek 293 ◽  
Trace Metal Ions ◽  
293 Cells ◽  
Better Than

Trace metal ions such as Zn2+, Fe2+, Cu2+, Mn2+, and Co2+ are required cofactors for many essential cellular enzymes, yet little is known about the mechanisms through which they enter into cells. We have shown previously that the widely expressed ion channel TRPM7 (LTRPC7, ChaK1, TRP-PLIK) functions as a Ca2+- and Mg2+-permeable cation channel, whose activity is regulated by intracellular Mg2+ and Mg2+·ATP and have designated native TRPM7-mediated currents as magnesium-nucleotide–regulated metal ion currents (MagNuM). Here we report that heterologously overexpressed TRPM7 in HEK-293 cells conducts a range of essential and toxic divalent metal ions with strong preference for Zn2+ and Ni2+, which both permeate TRPM7 up to four times better than Ca2+. Similarly, native MagNuM currents are also able to support Zn2+ entry. Furthermore, TRPM7 allows other essential metals such as Mn2+ and Co2+ to permeate, and permits significant entry of nonphysiologic or toxic metals such as Cd2+, Ba2+, and Sr2+. Equimolar replacement studies substituting 10 mM Ca2+ with the respective divalent ions reveal a unique permeation profile for TRPM7 with a permeability sequence of Zn2+ ≈ Ni2+ >> Ba2+ > Co2+ > Mg2+ ≥ Mn2+ ≥ Sr2+ ≥ Cd2+ ≥ Ca2+, while trivalent ions such as La3+ and Gd3+ are not measurably permeable. With the exception of Mg2+, which exerts strong negative feedback from the intracellular side of the pore, this sequence is faithfully maintained when isotonic solutions of these divalent cations are used. Fura-2 quenching experiments with Mn2+, Co2+, or Ni2+ suggest that these can be transported by TRPM7 in the presence of physiological levels of Ca2+ and Mg2+, suggesting that TRPM7 represents a novel ion-channel mechanism for cellular metal ion entry into vertebrate cells.

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