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F200S
site-directed mutagenesis, by expanding the size of the binding pocket the unspecific endonucleolytic activity is increased
F200S/W215S
site-directed mutagenesis, by expanding the size of the binding pocket the unspecific endonucleolytic activity is increased
V217G
site-directed mutagenesis, by expanding the size of the binding pocket the unspecific endonucleolytic activity is increased
W215S
site-directed mutagenesis, by expanding the size of the binding pocket the unspecific endonucleolytic activity is increased
D292A
decrease in activity
H308N
decrease in activity
R282A
loss of DNA binding
D190A
-
mutant enzyme is completely devoid of DNA repair activity and fails to rescue the genetic instability of Saccharomyces cerevisiae strain YW778
E68A
-
mutant retains both AP endonuclease and 3'-diesterase repair activity, lacks ability to protect Saccharomyces cerevisiae strain YW778 from spontaneous and drug-induced DNA lesions
H279A
-
mutant enzyme is completely devoid of DNA repair activity and fails to rescue the genetic instability of Saccharomyces cerevisiae strain YW778
T58C
-
Mutant enzyme is redox active. Site-directed mutagenesis is performed on the full-length zebrafish Ape pET15b vector using Stratagene Quikchange kit and verified by DNA sequencing. The T58C zApe vector is then transformed into Rosetta Escherichia coli (DE3). Stable ovarian cancer Skov-3X cells with the NFkappaB-Luc gene are cotransfected with plasmid pcDNA-mutant and a Renilla luciferase control reporter vector pRL-CMV using lipofectamine.
D179N
-
mild (10fold reduction) activity
D229N
-
reduction in activity
D44V
-
mutant retains glycosylase activity against oxidized pyrimidines, but the apparent rate constant for the lyase activity is significantly lower than the wild-type value
deltaQLY69-71
deletion of the entire loop, interferes with eversion of the damaged base from the helix, deficient in processing damaged DNA
E145Q
-
complete loss of catalytic activity
E261Q
-
catalytically inactive mutant, Glu261 is essential to catalysis
H109N
-
mild (10fold reduction) activity
H182N
-
reduction in activity
H216N
-
reduction in activity
H231N
-
reduction in activity
H69N
-
reduction in activity
Q261A
perturbs the conserved zinc finger, deficient in processing damaged DNA, can be reductively cross-linked to damaged base-containing DNA, deficient in regenerating free enzyme from the Nei-DNA covalent complex formed during the reaction
QLY69-71AAA
all amino acids in the QLY loop substituted with alanines, interferes with eversion of the damaged base from the helix, deficient in processing damaged DNA
R171A
perturbs the conserved zinc finger, deficient in processing damaged DNA
R184A
-
mutant enzyme maintains lyase activity but exhibits glycosylase specificity different from wild-type enzyme
R37A
-
strong positive effect on catalytic activity
R37A/E261Q
-
shows a three-fold decrease in AP-DNA binding affinity (Kd 340 nM)
S39L
-
mutant does not have significant glycosylase activity for oxidized pyrimidines, alhough it maintains AP lysae activity
Y72A
-
Tyr72 is important for the stability of the enzyme-substrate complex
Y72A/E261Q
-
double mutant shows a dissociation constant of Kd 97 nM
Y72F
-
Tyr72 is important for the stability of the enzyme-substrate complex
D179N
-
mild (10fold reduction) activity
-
E261Q
-
catalytically inactive mutant, Glu261 is essential to catalysis
-
H231N
-
reduction in activity
-
Y72A
-
Tyr72 is important for the stability of the enzyme-substrate complex
-
Y72F
-
Tyr72 is important for the stability of the enzyme-substrate complex
-
K267A
compared with wild-type, the affinity for the substrate analogue, 21 bp double-stranded DNA containing an apurinic/apyrimidinic-site analogue, is drastically decreased. The dissociation constant of the mutant is 0.38 microM
C118A
m6A demethylase activity similar to wild-type, about 25% decrease in apurinic/apyrimidinic lyase activity
C118A/C129A
about 50% decrease in m6A demethylase activity, about 20% decrease in apurinic/apyrimidinic lyase activity
C129A
about 90% decrease in m6A demethylase activity, about 30% decrease in apurinic/apyrimidinic lyase activity
C138A
-
stable Skov-3X cells with the NFB-Luc gene are cotransfected with plasmid pcDNA-mutant, mutant is redox active
C138S
-
repair activity is not affected
C138S/C99S
-
double mutants
C208A
-
stable Skov-3X cells with the NFB-Luc gene are cotransfected with plasmid pcDNA-mutant, mutant is redox active
C208S
-
repair activity is not affected
C296A
-
stable Skov-3X cells with the NFB-Luc gene are cotransfected with plasmid pcDNA-mutant, mutant is redox active
C296S
-
repair activity is not affected
C310A
-
stable Skov-3X cells with the NFB-Luc gene are cotransfected with plasmid pcDNA-mutant, mutant is redox active
C310S
-
repair activity is not affected
C65A/C93A
-
mutant form of TAT-APE1 is generated by insertion of full-length of APE1 C65A/C93A into pTAT-2.1
C65S/C99S
-
double mutants
C93A
-
stable Skov-3X cells with the NFB-Luc gene are cotransfected with plasmid pcDNA-mutant, mutant is redox active
C93S
-
repair activity is not affected
C99A
-
stable Skov-3X cells with the NFB-Luc gene are cotransfected with plasmid pcDNA-mutant, mutant is redox active
C99S
-
the mutant loses affinity for DNA and its activity is inhibited by 10 mM Mg2+, involvement of Cys99 in APE1's substrate binding and catalysis provides an example of involvement of a residue far from the active site
D210A
-
reduced single-turnover rate
D219A
replacement of Asp219 with alanine decreases both the DNA binding and the AP endonuclease activity of the enzyme compared to wild-type
D233A
no residual m6A demethylase activity, about 30% decrease in apurinic/apyrimidinic lyase activity
D283A
-
altered kinetic values
D283A/D308A
-
altered kinetic values compared to wild-type enzyme
D70R
-
binding affinity nearly identical with wild-type enzyme, reduced specific endonuclease activity, at Mg2+ concentrations below 1 mM the activity of the mutant sharply decreases
D90A
replacement of Asp90 with alanine results in decrease in endonuclease activity, but the DNA binding activity is preserved compared to wild-type
DELTA1-20
-
nuclear localization is significantly decreased
DELTA1-29
-
mutant enzyme retains activity against abasic sites in single-stranded DNA
DELTA1-36
-
mutant enzyme retains activity against abasic sites in single-stranded DNA
DELTA1-7
-
mutant shows nearly normal nuclear localization
DELTA1-7/E12A/D13A
-
nuclear localization is significantly decreased
DELTAP211
-
truncated hairpin, mutant protein is inactive
E12A/D13A
-
mutant shows nearly normal nuclear localization
E96Q/D210N
mutation (APE1 ED or simply ED) is created by site-specific mutagenesis of the pETApe1 plasmid, mutant cannot bind Mg2+ in the active site
F266A
-
30fold reduction in abasic dsDNA incision, complete loss of endoribonuclease activity against c-myc CRD
F319A
DNA glycosylase activity is reduced 52.6fold, activity towards abasic sites is reduced 1.5fold
H113A/C118A/C129A/H134A
no residual m6A demethylase activity, about 55% decrease in apurinic/apyrimidinic lyase activity
H231A
no residual m6A demethylase activity, about 20% decrease in apurinic/apyrimidinic lyase activity
H270A
DNA glycosylase activity is reduced 50fold, activity towards abasic sites is reduced 2.3fold
H270L
DNA glycosylase activity is reduced 71.4fold, activity towards abasic sites is reduced 3.7fold
H270R
DNA glycosylase activity is reduced 3.9fold, activity towards abasic sites is nearly identical to wild-type activity
H287A
no residual m6A demethylase activity, about 25% decrease in apurinic/apyrimidinic lyase activity
H309S
-
loss of abasic dsDNA incision
InsG212
-
extended hairpin, diminished recognition and binding to 5-hydroxycytosine-containing DNA
K133A
about 50% decrease in m6A demethylase activity, about 45% decrease in apurinic/apyrimidinic lyase activity
K212R
lower catalytic specificity than wild-type enzyme
K212S
-
mutant protein is inactive
K27Q
site-directed mutagenesis, chromatin-binding defective K27Q mutant, but the mutation of Lys27 in recombinant APE1 proteins does not affect acetylation by p300 at Lys6 in vitro
K299A/R301A
-
the mutation diminishes mitochondrial translocation of APE1
K3l/R4L/K6L/K7L
-
as the wild-type enzyme the mutant enzyme is predominantly localitzed in the nucleus
K6A/K7A
mutation targeting the gene regulation function. Mutant shows a diffuse nuclear staining, with a minimal localization in nucleoli. Mutant produces mitotic defects in Ape1-depleted cells, particularly promoting the formation of binucleated cells. Expression of the mutant protein increases the frequency of end-to-end fusions
K6R/K7R
-
no significant difference in nuclear localization between wild-type enzyme and mutant enzyme
N226A
increased cleavage rate at apurinic/apyrimidinic sites, ability to bind to damaged DNA decreases
N226A/N229A
ability of the mutant to bind damaged DNA is decreased, Vmax is almost identical to that of the wild-type enzyme
N229A
increased cleavage rate at apurinic/apyrimidinic sites, ability to bind to damaged DNA decreases
P211R
-
kinetic parameters similar to wild-type enzyme, decreased specificity of binding
Q315A
DNA glycosylase activity is reduced 1.6fold, activity towards abasic sites is increased 1.18fold
R156Q
-
100fold reduced DNA binding capacity
Y171A
-
enhancement by imidazole (in absence of tyrosine) is lower than that of wild-type enzyme. The ratio of turnover number to Km-value for DNA containing an abasic site is 50000fold lower than wild-type value at low salt concentration and 7500fold lower than wild-type value at high salt concentrations
Y171F/P173L/N174K
-
mutation results in 20000fold decrease in the reaction rate and reduced binding affinity
Y171H
-
mutant enzyme is not enhanceed by imidazole (in absence of tyrosine). The ratio of turnover number to Km-value for DNA containing an abasic site is 50000fold lower than wild-type value at low salt concentration
Y269A
-
the ratio of turnover number to Km-value for DNA containing an abasic site is 12.5fold lower than wild-type value at low salt concentration and 21.4fold lower than wild-type value at high salt concentrations
W199S
-
mutant is designed in order to investigate the involvement of the tryptophan residues in AP site recognition
W199S/W213S
-
mutant is designed in order to investigate the involvement of the tryptophan residues in AP site recognition
W213S
-
mutant is designed in order to investigate the involvement of the tryptophan residues in AP site recognition
A138D
-
production by site-directed mutagenesis
D335N
mutant retains its capacity to bind to AP sites in DNA, but loses its endonuclease activity
D251A
site-directed mutagenesis, the mutant shows a 3fold decline in the endonucleolytic cleavage activity
E57A
site-directed mutagenesis, the mutant shows a 2fold decline in the endonucleolytic cleavage activity
E57A/D251A
site-directed mutagenesis, the double mutant shows no 3'-5' exonuclease activity
F242S
site-directed mutagenesis, the mutant shows increased 3'-5' exonuclease catalytic efficiency compared to wild-type
W235S
site-directed mutagenesis, the mutant shows decreased 3'-5' exonuclease catalytic efficiency compared to wild-type
Y137S
site-directed mutagenesis, the mutant shows decreased 3'-5' exonuclease catalytic efficiency compared to wild-type
Y234S
site-directed mutagenesis, the mutant shows decreased 3'-5' exonuclease catalytic efficiency compared to wild-type
Y234S/W235S
site-directed mutagenesis, the mutant shows decreased 3'-5' exonuclease catalytic efficiency compared to wild-type
Y237S
site-directed mutagenesis, the mutant shows no 3'-5' exonuclease activity
D166N
mutant enzyme shows wild-type activity
K147Q
activity of mutant enzyme is severely attenuated
Q31E/D218S
almost no glycosylase activity
Q31S
significantly reduced glycosylase activity
W222A
almost no glycosylase activity
W222F
mutant is similarly active as wild type on 8-oxoguanine/C substrates
W69F
mutant is similarly active as wild type on 8-oxoguanine/C substrates
D166N
-
mutant enzyme shows wild-type activity
-
D172Q
-
binds to 8-oxoguanine containing single-stranded DNA more tightly than double-stranded DNA containing a 8-oxoguanine/cytosine base pair
-
K140Q
-
inactive mutant enzyme
-
K147Q
-
activity of mutant enzyme is severely attenuated
-
W222A
-
almost no glycosylase activity
-
W222F
-
mutant is similarly active as wild type on 8-oxoguanine/C substrates
-
W69F
-
mutant is similarly active as wild type on 8-oxoguanine/C substrates
-
Y127F
-
decreased endonuclease activity, Tyr 127 is essential to the binding and stabilization of the Fe3+ ion, mutation do not affect DNA binding
E261A
the mutant retains some activity
H70A/H110A
the mutant retains residual enzyme activity
H70A/H110A/E261A
the mutant completely loses the activity
C65A
-
redox deficient-DNA repair competent (adenovirus infected)
N226A/R177A
-
DNA repair-deficient-redox competent Ape1 does not protect rat neurons from cisplatin toxicity (adenovirus infected)
K364R
-
ntg1, localized to both nuclei and mitochondria
E233S
site-directed mutagenesis
Y105A
site-directed mutagenesis
E233S
-
site-directed mutagenesis
-
Y105A
-
site-directed mutagenesis
-
W200S
-
mutant is designed in order to investigate the involvement of the tryptophan residues in AP site recognition
W200S/W214S
-
mutant is designed in order to investigate the involvement of the tryptophan residues in AP site recognition
W214S
-
mutant is designed in order to investigate the involvement of the tryptophan residues in AP site recognition
C65A
-
redox deficient mutant
C65A
-
site-directed mutagenesis of a pET28A vector encoding human Ape1 (40-318) is performed, mutant is redox inactive
C65S
-
repair activity is not affected
C65S
mutation targeting the Ref-1 redox functions. Mutant form is detected in cytoplasmic vesicles indicating altered turnover. The endonuclease domain of Ape1 is required for successful mitotic progression
D210N
-
catalytically inactive
D210N
-
mutant enzyme is catalytically inactive against abasic sites in double-stranded DNA and single-stranded DNA
D210N
-
loss of abasic dsDNA incision, complete loss of endoribonuclease activity against c-myc CRD
D283N
-
loss of abasic dsDNA incision, no change in endoribonuclease activity against c-myc CRD
D283N
-
retaines endoribonuclease and abasic single-stranded RNA cleavage activities, with concurrent loss of apurinic/apyrimidinic site cleavage activities on double-stranded DNA and single-stranded DNA
D308A
-
altered kinetic values compared to wild-type enzyme
D308A
-
1.4fold reduction in abasic dsDNA incision, complete loss of endoribonuclease activity against c-myc CRD
D308A
replacement of Asp308 with alanine results in decrease in endonuclease activity, but the DNA binding activity is preserved compared to wild-type
D70A
-
mutant increases capacity to remove 3'-blocking ends in vitro
D70A
-
6.7fold reduction in abasic dsDNA incision, complete loss of endoribonuclease activity against c-myc CRD
E96A
-
exonuclease deficient mutant
E96A
-
APE-1 mutant displays a significantly reduced DNA-repair activity when compared with the wild-type protein
E96A
-
the mutant does not exhibit any nuclease activity against 88-nt RNA substrate
E96A
-
no reduction in abasic dsDNA incision, complete loss of endoribonuclease activity against c-myc CRD
E96A
replacement of Glu96 with alanine results in decrease in endonuclease activity, but the DNA binding activity is preserved compared to wild-type
E96Q
-
binding affinity nearly identical with wild-type enzyme, reduced specific endonuclease activity
E96Q
-
exonuclease deficient mutant
E96Q
the mutant is expressed well in both TB and autoinducing media, the E96Q mutation prevents Mg2+ binding at this site
H309N
-
altered kinetic values compared to wild-type enzyme
H309N
-
reduced single-turnover rate
H309N
-
the mutant does not exhibit any nuclease activity against 88-nt RNA substrate
H309N
site-directed mutagenesis, the mutant still binds to chromatin and gets acetylated
N212A
mutation targeting the endonuclease function. Mutant shows mainly nuclear staining, with a focal accumulation in nucleoli. Expression of the mutant protein increases the frequency of end-to-end fusions
N212A
simulations with substitution of Asn212 by Ala show a Mg2+-ion coordination comparable to that of the wild-type
N68A
-
binding affinity nearly identical with wild-type enzyme, reduced specific endonuclease activity, at Mg2+ concentrations below 1 mM the activity of the mutant sharply decreases
N68A
-
loss of abasic dsDNA incision, complete loss of endoribonuclease activity against c-myc CRD
R177A
APE1's high affinity for DNA single-strand breaks requires Arg177, poly(ADP-ribose)polymerase 1, PARP1, activation is not suppressed by the mutant
R177A
he turnover number of the endonuclease reaction catalyzed by APE1 increases compared to wild-type when Arg177 is replaced by alanine
Y128A
-
100fold reduced DNA binding capacity
Y128A
-
the ratio of turnover number to Km-value for DNA containing an abasic site is 33fold lower than wild-type value at low salt concentration and 7.5fold lower than wild-type value at high salt concentrations
Y171F
-
the ratio of turnover number to Km-value for DNA containing an abasic site is 25000fold lower than wild-type value at low salt concentration
Y171F
-
loss of abasic dsDNA incision, complete loss of endoribonuclease activity against c-myc CRD
K129S
inactive mutant enzyme
K129S
-
inactive mutant enzyme
-
D172N
inactive mutant enzyme
D172N
binds to 8-oxoguanine containing single-stranded DNA more tightly than double-stranded DNA containing a 8-oxoguanine/cytosine base pair
D172Q
binds to 8-oxoguanine containing single-stranded DNA more tightly than double-stranded DNA containing a 8-oxoguanine/cytosine base pair
D172Q
no detectable glycosylase activity
K140Q
inactive mutant enzyme
K140Q
binds to 8-oxoguanine containing single-stranded DNA more tightly than double-stranded DNA containing a 8-oxoguanine/cytosine base pair
D172N
-
binds to 8-oxoguanine containing single-stranded DNA more tightly than double-stranded DNA containing a 8-oxoguanine/cytosine base pair
-
D172N
-
inactive mutant enzyme
-
H83A
site-directed mutagenesis, the mutation decrease the AP endonuclease activity of Apn1 owing to weak coordination of Zn2+ ions involved in enzymatic catalysis, suppressed enzymatic activity of H83A Apn1 results from the reduced number of active site Zn2+ ions. Analysis of kinetics of recognition, binding, and incision of DNA substrates of the H83A Apn1 mutant. Substitution of His83 with Ala influences catalytic complex formation and further incision of the damaged DNA strand. The H83A Apn1 catalysis depends not only on the location of the mismatch relative to the abasic site in DNA, but also on the nature of damage. H83A Apn1 appears to cleave substrates AP(2-aPu) and F(2-aPu) in several stages (F is tetrahydrofuran). Minimal kinetic mechanism of abasic site cleavage by H83A Apn1, molecular dynamics of H83A Apn1, overview. Molecular dynamics simulations of the H83A Apn1 structure containing the two Zn2+ ions reveal an insignificant movement of Zn2 relative to DNA and amino acid residues involved in Zn2 coordination. Structure of enzyme mutant H83A Apn1-substrate DNA complex with three Zn2+ ions containing Zn2+ ions per molecule of mutant enzyme, overview
H83A
site-directed mutagenesis, the mutation decrease the AP endonuclease activity of Apn1 owing to weak coordination of Zn2+ ions involved in enzymatic catalysis, suppressed enzymatic activity of H83A Apn1 results from the reduced number of active site Zn2+ ions. 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
H83A
-
site-directed mutagenesis, the mutation decrease the AP endonuclease activity of Apn1 owing to weak coordination of Zn2+ ions involved in enzymatic catalysis, suppressed enzymatic activity of H83A Apn1 results from the reduced number of active site Zn2+ ions. Analysis of kinetics of recognition, binding, and incision of DNA substrates of the H83A Apn1 mutant. Substitution of His83 with Ala influences catalytic complex formation and further incision of the damaged DNA strand. The H83A Apn1 catalysis depends not only on the location of the mismatch relative to the abasic site in DNA, but also on the nature of damage. H83A Apn1 appears to cleave substrates AP(2-aPu) and F(2-aPu) in several stages (F is tetrahydrofuran). Minimal kinetic mechanism of abasic site cleavage by H83A Apn1, molecular dynamics of H83A Apn1, overview. Molecular dynamics simulations of the H83A Apn1 structure containing the two Zn2+ ions reveal an insignificant movement of Zn2 relative to DNA and amino acid residues involved in Zn2 coordination. Structure of enzyme mutant H83A Apn1-substrate DNA complex with three Zn2+ ions containing Zn2+ ions per molecule of mutant enzyme, overview
-
H83A
-
site-directed mutagenesis, the mutation decrease the AP endonuclease activity of Apn1 owing to weak coordination of Zn2+ ions involved in enzymatic catalysis, suppressed enzymatic activity of H83A Apn1 results from the reduced number of active site Zn2+ ions. 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
protein with two additional amino acid residues and a truncated protein with deletion of 22 residues at the amino-terminus are equally active
additional information
-
protein with two additional amino acid residues and a truncated protein with deletion of 22 residues at the amino-terminus are equally active
additional information
APE1 lacking the first 34 amino acids at the Nterminus, unlike wild-type enzyme, is unable to form cross-links with BS-AP DNAs that testifies to the involvement of disordered N-terminal extension, which is enriched in lysine residues, in the interaction with AP sites.
additional information
-
APE1 lacking the first 34 amino acids at the Nterminus, unlike wild-type enzyme, is unable to form cross-links with BS-AP DNAs that testifies to the involvement of disordered N-terminal extension, which is enriched in lysine residues, in the interaction with AP sites.
additional information
apurinic/apyrimidinic endonuclease 1-siRNA-mediated downregulation in osteosarcoma cells inhibits expression of TGFbeta1. Apurinic/apyrimidinic endonuclease 1-siRNA inhibits the capability to enhance HUVEC migration and tube formation of tumor cells through the TGFbeta/Smad3 signaling pathway. Tumor angiogenesis and growth in xenografts are suppressed by APE1-siRNA
additional information
-
apurinic/apyrimidinic endonuclease 1-siRNA-mediated downregulation in osteosarcoma cells inhibits expression of TGFbeta1. Apurinic/apyrimidinic endonuclease 1-siRNA inhibits the capability to enhance HUVEC migration and tube formation of tumor cells through the TGFbeta/Smad3 signaling pathway. Tumor angiogenesis and growth in xenografts are suppressed by APE1-siRNA
additional information
construction of a redox-deficient truncated APE1 protein lacking the first N-terminal 61 amino acid residues (APE1-NDELTA61) the mutant cannot stimulate DNA glycosylase activities of OGG1, MBD4, and ANPG on duplex DNA substrates in contrast to the wild-type enzyme
additional information
-
construction of a redox-deficient truncated APE1 protein lacking the first N-terminal 61 amino acid residues (APE1-NDELTA61) the mutant cannot stimulate DNA glycosylase activities of OGG1, MBD4, and ANPG on duplex DNA substrates in contrast to the wild-type enzyme
additional information
downregulation of endogenous APE1 levels in HEK293T cells using small interfering RNA (siRNA). Mutant phenotypes, overview
additional information
-
downregulation of endogenous APE1 levels in HEK293T cells using small interfering RNA (siRNA). Mutant phenotypes, overview
additional information
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deletion mutant delta369 and delta491, the C-terminal deletion does not affect the AP endonuclease activity, but the protein is defective in the removal of AP sites in vivo