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Cu2+
activates at 0.01 mM, inhibits at 0.1 mM
Fe
-
pro-inflammatory activity of iron in the lung injury, at least in part, because of its induction of APE/Ref-1
MgCl2
-
tetrahydrofuran*G incision is efficiently catalyzed at 0.001 mM Mg2+, 5 mM MgCl2 are required for optimal AP endonuclease activity
Na+
-
65 mM included in assay medium
Sm2+
-
the divalent metal ion soaked with the protein crystals is found specifically to associate with the glutamate residue
Ca2+
-
stimulates
Ca2+
presence of 1 or 5 mM Ca2+ causes much less efficient stimulation of HpXth's AP site cleavage activity as compared to Mg2+ or Mn2+
Ca2+
-
optimal concentration at 5-30 mM, only 50% activity at 40 mM
Co2+
-
CoCl2 (500 microM) is essential for AP endonuclease assay. Effects of Co on APE/Ref-1 are concentration dependent
Co2+
-
0.1-1.0 mM, strong stimulation of all DNA repair activities
Fe2+
EndoIV contains two Fe2+ ions and one Zn2+ ion, crystallography data. Fe2+(1) is coordinated by Glu145, Asp179, His214, Glu259, and Fe2+(2) is coordinated by His69, His110 and Glu145
Fe2+
activates at 0.01 mM, inhibits at 0.1 mM
Fe2+
-
Fe2+ is able to support the incision activity of the enzyme at excess protein to DNA ratios of at least 6:1, Mg2+ and Fe2+ compete for the same metal-binding site
Fe2+
-
0.1-1.0 mM, strong stimulation of all DNA repair activities
Iron
-
iron-sulfur protein
Iron
-
native enzyme contains a single [4Fe-4S] cluster in the 2+ oxidation state with a net spin of zero
Iron
-
[4Fe-4S] cluster is not directly involved in catalytic mechanism and therefore has most likely a structural role
Iron
-
iron-sulfur protein
Iron
-
Scr2 but not Scr1 is an iron-sulfur protein
K+
-
both MgCl2 and KCl strongly influence the efficiency of Ape1 as an ssDNA or dsDNA AP endonuclease
K+
-
K+ significantly stimulates recombinant APE1 activity at 20 mM by approximately 1.3fold (compared with the absence of K+)
K+
-
25 mM included in assay medium. DNA structures with a nick and DNArec used for photoaffinity modification are substrates for APE1 3'-5'-exonuclease activity, that is more efficient at decreased salt concentrations.
K+
-
K+ significantly stimulates recombinant APE1 activity at 20 mM by approximately 1.3fold (compared with the absence of K+)
KCl
-
stimulates
KCl
-
optimal concentration: 25-50 mM
KCl
-
optimal concentration: 0.05-0.1 M, activity against apurinic and apyrimidinic sites, 50% inhibition at 0.02 and 0.12 M
KCl
-
optimal concentration: 0.10 M, activity against OsO4-sites, 50% inhibition at 0.05 and 0.15 M
KCl
-
optimal concentration: 125 mM
KCl
-
maximal AP endonuclease activity at 25-200 mM, nucleotide incision repair activity decreases dramatically above 50 mM
KCl
optimum concentration 50-100 mM
KCl
-
optimal concentration: 100 mM
KCl
-
optimal concentration: 50 mM,90% inhibition at 200 mM
KCl
-
enzyme form A: optimal activity in 20 mM NaCl or KCl, enzyme form B: more active without salt
KCl
optimum concentration 50-100 mM
KCl
-
slight stimulation at 10 to 30 mM, inhibition above 100 mM
KCl
-
optimum concentration 20 mM
Mg2+
-
the optimized buffer used for the endonuclease assay contains 5 mM MgCl2
Mg2+
-
optimal concentration: 20 mM
Mg2+
-
or Mn2+, absolutely required
Mg2+
-
Mg2+ or Mn2+ required, Mg2+ better than Mn2+
Mg2+
-
optimal concentration: 10 mM
Mg2+
-
Mg2+ or Mn2+ required
Mg2+
required, activates, best at 5 mM
Mg2+
-
optimal concentration: 3 mM
Mg2+
metal-dependent enzyme
Mg2+
-
repair of 5-OH-C opposite guanine is stimulated 2-fold with increasing concentrations, 5-OH-C paired with adenine is poorly repaired with increasing Mg2+ concentrations, no incision of 5-OH-C opposite adenine above 15 mM Mg2+
Mg2+
-
endonuclease B has an absolute requirement for Mg2+
Mg2+
-
endonuclease A does not require Mg2+ for full activity
Mg2+
-
activity against abasic sites in single-stranded DNA
Mg2+
APE1 binds to AP sites in the absence of Mg2+, a condition in which APE1 does not have endonuclease activity
Mg2+
-
included in assay medium
Mg2+
-
5 mM included in assay medium, required divalent cation
Mg2+
-
APE1 reaches a maximum of 3'-phosphoglycolate excision activity at Mg2+ concentrations around 2.5 mM, APE1 mutant D70A reaches maximum activity at higher metal concentrations. In case of a THF-containing oligonucleotide as substrate, APE1 exhibits the highest AP endonuclease activity at 5 mM of metal while the specific activity of APE1 mutant D70A did not reach a maximum until 40 mM of Mg2+ is added to the reaction.
Mg2+
binds to APE1 and a functional APE1-substrate DNA complex with an overall stoichiometry of one Mg2+ per mole of APE1, the chemistry central to the function of APE1 is water activated by a Mg2+ ion, Mg2+ binding is an absolute requirement for the endonucleolytic activity
Mg2+
-
In presence of Mg2+, ATP has complex effects on Ape1 cleavage activity. Endo- and exonuclease activity of Ape1 is found to be influenced by the MgCl2 concentration, with optimal exonuclease activity at low MgCl2 concentration (0.1-2 mM) and optimal endonuclease activity at high MgCl2 concentration (10-15 mM).
Mg2+
-
Mg2+, with potential binding sites A and B, binds at the B site of wild-type APE1-substrate complex and moves to the A site after cleavage occurs
Mg2+
-
ion concentrations ranging from 0.2 to 2 mM Mg2+ promotes catalysis, APE1 is enhanced approximately 2fold (compared with the absence of Mg2+) in the presence of 2 mM Mg2+
Mg2+
-
required for APE1-catalyzed endonuclease activity
Mg2+
-
both the endoribonuclease and the ssRNA apurinic/apyrimidinic site cleavage activities of wild-type APE1 are present in the absence of Mg2+, while ssDNA apurinic/apyrimidinic site cleavage requires Mg2+, optimally at 0.5-2.0 mM
Mg2+
required. Increasing the Mg2+ concentration alters the ratio of turns to beta-strands, and this change may be associated with the conformational changes required to achieve an active state
Mg2+
activates, Mg2+ or Mn2+ is required for endonuclease and exonuclease activity of APE1. Mg2+ is required for both binding of the protein with DNA and cleavage of phosphodiester bond catalyzed by APE1. Depending on the Mg2+ concentration, the limiting stage of the process can change
Mg2+
activates, required and involved in catalytic mechanism, Mg2+ ions stabilize the protein structure and the enzyme-substrate complex. Analysis of enzyme-substrate complexes with bound Mg2+
Mg2+
-
LMAP mutant A138D reaches a maximum of 3'-PG excision activity at Mg2+ concentrations around 2.5 mM, wild-type enzyme reaches maximum activity at higher metal concentrations. In case of a THF-containing oligonucleotide as substrate, the parasite enzymes LMAP and LMAPA138D, obtains the activity peaks at different magnesium concentrations (10 and 2.5 mM).
Mg2+
-
optimal concentration: 4-5 mM
Mg2+
-
optimal concentration: 1-5 mM
Mg2+
-
2 mM included in assay medium
Mg2+
required. Increasing the Mg2+ concentration alters the ratio of turns to beta-strands, and this change may be associated with the conformational changes required to achieve an active state
Mg2+
required, MtbXthA is inactive in AP site incision assays in the absence of Mg2+, while increasing activity is observed with increasing Mg2+ concentration between 1-10 mM. The protein exhibits maximal incision activity, 60%, when no NaCl is included in the buffer
Mg2+
-
the standard reaction buffer used for the nuclease assay is supplemented with 15 mM MgCl2
Mg2+
maximally stimulated at 10 mM
Mg2+
-
Mg2+ or Mn2+ required
Mg2+
-
optimal concentration: 5-10 mM
Mg2+
-
ion concentrations ranging from 0.2 to 2 mM Mg2+ promotes catalysis, APE1 is enhanced approximately 2fold (compared with the absence of Mg2+) in the presence of 2 mM Mg2+
Mg2+
-
Mg2+ or Mn2+ required
Mg2+
-
optimal concentration: 4 mM for endonuclease D1 and D2
Mg2+
-
optimal concentration: 8 mM, endonuclease D3
Mg2+
-
optimal concentration: 6 mM, endonuclease E
Mg2+
-
optimal concentration: 2 mM (endonuclease D4)
Mg2+
-
optimal concentration: 2.5-30 mM
Mg2+
APN is active in presence and in absence of Mg2+
Mn2+
-
stimulates
Mn2+
-
or Mg2+, absolutely required
Mn2+
-
can only partially replace Mg2+
Mn2+
-
Mn2+ or Mg2+ required
Mn2+
-
Mn2+ or Mg2+ required
Mn2+
-
divalent metal content
Mn2+
-
partial stimulation
Mn2+
-
Glu in amino acid position 96 binds to the divalent cation
Mn2+
activates, Mg2+ or Mn2+ is required for endonuclease and exonuclease activity of APE1
Mn2+
-
In order to cleave phosphodiester bonds in the course of endonuclease and exonuclease reactions catalyzed by APE1, Mg2+ or Mn2+ is needed
Mn2+
maximally stimulated at 10 mM
Mn2+
-
Mn2+ or Mg2+ required
Mn2+
-
Mn2+ or Mg2+ required
Mn2+
-
0.1-1.0 mM, strong stimulation of all DNA repair activities
NaCl
-
50 mM: slight stimulation, 500 mM: complete inhibition
NaCl
-
optimal concentration: 25-50 mM
NaCl
-
optimal concentration: 50 mM
NaCl
-
enzyme form A: optimal activity in 20 mM NaCl or KCl, enzyme form B: more active without salt
NaCl
MtbXthA exhibits an increase in AP site incision activity in a salt-dependent manner. Optimum conditions are 2 mM MgCl2 and 150 mM NaCl with 75% incision activity. Addition of more than 200 mM salt, strongly inhibits the endonuclease activity
NaCl
-
slight stimulation at 10 to 30 mM, inhibition above 100 mM
NaCl
-
150 mM, higher concentrations inhibit
NaCl
-
50 mM, 50% stimulation
Ni2+
activates at 0.01 mM, inhibits at 0.1 mM
Zn2+
-
the optimized buffer used for the exonuclease assay contains 5 mM ZnCl2
Zn2+
-
Divalent metal content, Zn3-site mutations result in major activity loss, whereas Zn1 and Zn2 ligand mutations cause low to severe loss of catalytic efficiency. In the DNA-free wild-type enzyme structure, two metal ions (Zn1 and Zn2) are partially buried from solvent and bind a bridging hydroxide anion. The third metal ion (Zn3) is mostly solvent accessible, is distant from Zn1 and Zn2 and ligates a tightly bound water molecule to complete its coordination shell
Zn2+
EndoIV contains two Fe2+ ions and one Zn2+ ion, crystallography data. The Zn2+ ion is coordinated by His182, Asp227, His229
Zn2+
activates at 0.01 mM, inhibits at 0.1 mM
Zn2+
-
stimulation half as effective as with Mg2+
Zn2+
His83 coordinates one of three Zn2+ ions in Apn1's active site, structure comparisons. Structure of enzyme mutant H83A Apn1-substrate DNA complex with three Zn2+ ions containing Zn2+ ions per molecule of mutant enzyme, overview. Zn2+ ions are involved in catalysis
Zn2+
required for catalysis, molecular dynamics. The active site of H83A Apn1 contains only two Zn2+ ions, with their positions being changed versus a trinuclear Zn2+ cluster of wild-type Apn1
additional information
-
Mn2+ has no effect
additional information
-
equal or higher activity for Zn2+ or Mn2+- containing Endo IV suggests that one site may favor Mn2+ over Zn2+
additional information
in the presence of 1 or 5 mM Mg2+ or Mn2+, the purified recombinant His-tagged HpXth protein exerts an efficient AP site cleavage activity by generating fast-migrating 10mer cleavage fragments. But higher concentrations of Mg2+ or Mn2+ (10-20 mM) result in a strong decrease in the HpXth-catalyzed AP site cleavage
additional information
-
in the presence of 1 or 5 mM Mg2+ or Mn2+, the purified recombinant His-tagged HpXth protein exerts an efficient AP site cleavage activity by generating fast-migrating 10mer cleavage fragments. But higher concentrations of Mg2+ or Mn2+ (10-20 mM) result in a strong decrease in the HpXth-catalyzed AP site cleavage
additional information
effects of monovalent (K+) and divalent (Mg2+, Mn2+, Ca2+, Zn2+, Cu2+, and Ni2+) metal ions on DNA binding and catalytic stages, circular dichroism spectra and calculation of the contact area between APE1 and DNA, overview. The first step of substrate binding (corresponding to formation of a primary enzyme-substrate complex) does not depend on the concentration (0.05-5.0 mM) or the nature of divalent metal ions. In contrast, the initial DNA binding efficiency significantly decreases at a high concentration (5-250 mM) of monovalent K+ ions, indicating the involvement of electrostatic interactions in this stage. The enzymatic activity of APE1 is increased in the ascending order Zn2+, Ni2+, Mn2+, and Mg2+
additional information
-
effects of monovalent (K+) and divalent (Mg2+, Mn2+, Ca2+, Zn2+, Cu2+, and Ni2+) metal ions on DNA binding and catalytic stages, circular dichroism spectra and calculation of the contact area between APE1 and DNA, overview. The first step of substrate binding (corresponding to formation of a primary enzyme-substrate complex) does not depend on the concentration (0.05-5.0 mM) or the nature of divalent metal ions. In contrast, the initial DNA binding efficiency significantly decreases at a high concentration (5-250 mM) of monovalent K+ ions, indicating the involvement of electrostatic interactions in this stage. The enzymatic activity of APE1 is increased in the ascending order Zn2+, Ni2+, Mn2+, and Mg2+
additional information
there is only one bivalent metal ion in the APE1 active site present in the crystal formed at pH 4.6. In the structure produced at pH 7.5, i.e. under conditions optimal for endonuclease activity, two metal ions are located in the active site of the enzyme. MgCl2 concentration in the range 0.5-2.0 mM and low (50 mM or below) concentration of KCl are optimal for cleavage of single-stranded DNA with an AP site, while the endonuclease activity towards the double-stranded AP-DNA is the highest at 10 mM MgCl2 and 50 mM KCl or 2 mM MgCl2 and 200 mM KCl. Co2+ and Ni2+ do not affect APE1 activity
additional information
-
there is only one bivalent metal ion in the APE1 active site present in the crystal formed at pH 4.6. In the structure produced at pH 7.5, i.e. under conditions optimal for endonuclease activity, two metal ions are located in the active site of the enzyme. MgCl2 concentration in the range 0.5-2.0 mM and low (50 mM or below) concentration of KCl are optimal for cleavage of single-stranded DNA with an AP site, while the endonuclease activity towards the double-stranded AP-DNA is the highest at 10 mM MgCl2 and 50 mM KCl or 2 mM MgCl2 and 200 mM KCl. Co2+ and Ni2+ do not affect APE1 activity
additional information
-
cleavage of abasic DNA by UL30 occurs in the presence of EDTA and is independent of Mg2+, UL30 is 10fold less active
additional information
-
presence of divalent cations does not stimulate the activity
additional information
the highly conserved catalytic site in AP endonucleases consists of residues involved in the binding of metal ions. MtbXthA exhibits moderate 3'-5' exonuclease activity at low ionic environment
additional information
-
the highly conserved catalytic site in AP endonucleases consists of residues involved in the binding of metal ions. MtbXthA exhibits moderate 3'-5' exonuclease activity at low ionic environment
additional information
the nucleotide incision repair of the enzyme in presence of Mg2+ is unaltered, Mg2+ does not affect yeast Ape1 activity