ape1  (New England Biolabs)


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    Name:
    APE 1
    Description:
    APE 1 5 000 units
    Catalog Number:
    m0282l
    Price:
    293
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    5 000 units
    Category:
    Other Endonucleases
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    Structured Review

    New England Biolabs ape1
    APE 1
    APE 1 5 000 units
    https://www.bioz.com/result/ape1/product/New England Biolabs
    Average 99 stars, based on 57 article reviews
    Price from $9.99 to $1999.99
    ape1 - by Bioz Stars, 2020-10
    99/100 stars

    Images

    1) Product Images from "Alzheimer’s Disease Associated Polymorphisms in Human OGG1 Alter Catalytic Activity and Sensitize Cells to DNA Damage"

    Article Title: Alzheimer’s Disease Associated Polymorphisms in Human OGG1 Alter Catalytic Activity and Sensitize Cells to DNA Damage

    Journal: Free radical biology & medicine

    doi: 10.1016/j.freeradbiomed.2013.05.010

    Decreased AP lyase activity and differential stimulation of polymorphic OGG1s by APE1 (A) OGG1 recombinant proteins (64nM) were incubated at 37°C for 15 minutes in the presence (+) or absence (−) of 45pM APE1, along with a 5′-end-labeled oligonucleotide duplex containing an 8-oxoG lesion. The cleavage products were analyzed on a 15% polyacrylamide gel containing 7M Urea and imaged on a phosphorimager. A representative experiment is shown. (B) The histogram represents the mean ± S.E.M. from five independent experiments. The incision value was calculated by taking the amount of cleaved substrate (lower band) normalized to the amount of cleaved + uncleaved substrate (lower + upper bands) . *p
    Figure Legend Snippet: Decreased AP lyase activity and differential stimulation of polymorphic OGG1s by APE1 (A) OGG1 recombinant proteins (64nM) were incubated at 37°C for 15 minutes in the presence (+) or absence (−) of 45pM APE1, along with a 5′-end-labeled oligonucleotide duplex containing an 8-oxoG lesion. The cleavage products were analyzed on a 15% polyacrylamide gel containing 7M Urea and imaged on a phosphorimager. A representative experiment is shown. (B) The histogram represents the mean ± S.E.M. from five independent experiments. The incision value was calculated by taking the amount of cleaved substrate (lower band) normalized to the amount of cleaved + uncleaved substrate (lower + upper bands) . *p

    Techniques Used: Activity Assay, Recombinant, Incubation, Labeling

    2) Product Images from "Alzheimer’s Disease Associated Polymorphisms in Human OGG1 Alter Catalytic Activity and Sensitize Cells to DNA Damage"

    Article Title: Alzheimer’s Disease Associated Polymorphisms in Human OGG1 Alter Catalytic Activity and Sensitize Cells to DNA Damage

    Journal: Free radical biology & medicine

    doi: 10.1016/j.freeradbiomed.2013.05.010

    Decreased AP lyase activity and differential stimulation of polymorphic OGG1s by APE1 (A) OGG1 recombinant proteins (64nM) were incubated at 37°C for 15 minutes in the presence (+) or absence (−) of 45pM APE1, along with a 5′-end-labeled oligonucleotide duplex containing an 8-oxoG lesion. The cleavage products were analyzed on a 15% polyacrylamide gel containing 7M Urea and imaged on a phosphorimager. A representative experiment is shown. (B) The histogram represents the mean ± S.E.M. from five independent experiments. The incision value was calculated by taking the amount of cleaved substrate (lower band) normalized to the amount of cleaved + uncleaved substrate (lower + upper bands) . *p
    Figure Legend Snippet: Decreased AP lyase activity and differential stimulation of polymorphic OGG1s by APE1 (A) OGG1 recombinant proteins (64nM) were incubated at 37°C for 15 minutes in the presence (+) or absence (−) of 45pM APE1, along with a 5′-end-labeled oligonucleotide duplex containing an 8-oxoG lesion. The cleavage products were analyzed on a 15% polyacrylamide gel containing 7M Urea and imaged on a phosphorimager. A representative experiment is shown. (B) The histogram represents the mean ± S.E.M. from five independent experiments. The incision value was calculated by taking the amount of cleaved substrate (lower band) normalized to the amount of cleaved + uncleaved substrate (lower + upper bands) . *p

    Techniques Used: Activity Assay, Recombinant, Incubation, Labeling

    3) Product Images from "Alzheimer’s Disease Associated Polymorphisms in Human OGG1 Alter Catalytic Activity and Sensitize Cells to DNA Damage"

    Article Title: Alzheimer’s Disease Associated Polymorphisms in Human OGG1 Alter Catalytic Activity and Sensitize Cells to DNA Damage

    Journal: Free radical biology & medicine

    doi: 10.1016/j.freeradbiomed.2013.05.010

    Decreased AP lyase activity and differential stimulation of polymorphic OGG1s by APE1 (A) OGG1 recombinant proteins (64nM) were incubated at 37°C for 15 minutes in the presence (+) or absence (−) of 45pM APE1, along with a 5′-end-labeled oligonucleotide duplex containing an 8-oxoG lesion. The cleavage products were analyzed on a 15% polyacrylamide gel containing 7M Urea and imaged on a phosphorimager. A representative experiment is shown. (B) The histogram represents the mean ± S.E.M. from five independent experiments. The incision value was calculated by taking the amount of cleaved substrate (lower band) normalized to the amount of cleaved + uncleaved substrate (lower + upper bands) . *p
    Figure Legend Snippet: Decreased AP lyase activity and differential stimulation of polymorphic OGG1s by APE1 (A) OGG1 recombinant proteins (64nM) were incubated at 37°C for 15 minutes in the presence (+) or absence (−) of 45pM APE1, along with a 5′-end-labeled oligonucleotide duplex containing an 8-oxoG lesion. The cleavage products were analyzed on a 15% polyacrylamide gel containing 7M Urea and imaged on a phosphorimager. A representative experiment is shown. (B) The histogram represents the mean ± S.E.M. from five independent experiments. The incision value was calculated by taking the amount of cleaved substrate (lower band) normalized to the amount of cleaved + uncleaved substrate (lower + upper bands) . *p

    Techniques Used: Activity Assay, Recombinant, Incubation, Labeling

    4) Product Images from "Alzheimer’s Disease Associated Polymorphisms in Human OGG1 Alter Catalytic Activity and Sensitize Cells to DNA Damage"

    Article Title: Alzheimer’s Disease Associated Polymorphisms in Human OGG1 Alter Catalytic Activity and Sensitize Cells to DNA Damage

    Journal: Free radical biology & medicine

    doi: 10.1016/j.freeradbiomed.2013.05.010

    Decreased AP lyase activity and differential stimulation of polymorphic OGG1s by APE1 (A) OGG1 recombinant proteins (64nM) were incubated at 37°C for 15 minutes in the presence (+) or absence (−) of 45pM APE1, along with a 5′-end-labeled oligonucleotide duplex containing an 8-oxoG lesion. The cleavage products were analyzed on a 15% polyacrylamide gel containing 7M Urea and imaged on a phosphorimager. A representative experiment is shown. (B) The histogram represents the mean ± S.E.M. from five independent experiments. The incision value was calculated by taking the amount of cleaved substrate (lower band) normalized to the amount of cleaved + uncleaved substrate (lower + upper bands) . *p
    Figure Legend Snippet: Decreased AP lyase activity and differential stimulation of polymorphic OGG1s by APE1 (A) OGG1 recombinant proteins (64nM) were incubated at 37°C for 15 minutes in the presence (+) or absence (−) of 45pM APE1, along with a 5′-end-labeled oligonucleotide duplex containing an 8-oxoG lesion. The cleavage products were analyzed on a 15% polyacrylamide gel containing 7M Urea and imaged on a phosphorimager. A representative experiment is shown. (B) The histogram represents the mean ± S.E.M. from five independent experiments. The incision value was calculated by taking the amount of cleaved substrate (lower band) normalized to the amount of cleaved + uncleaved substrate (lower + upper bands) . *p

    Techniques Used: Activity Assay, Recombinant, Incubation, Labeling

    5) Product Images from "Influence of Oxidized Purine Processing on Strand Directionality of Mismatch Repair *"

    Article Title: Influence of Oxidized Purine Processing on Strand Directionality of Mismatch Repair *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.629907

    MYH is active in HCT116 extracts and addresses G O /A but not A O /G or A O /C mispairs. A , nicking assay. The closed-circular homoduplex or the G O /A, A O /G, or A O /C substrates were incubated with recombinant, purified MYH-GST and APE1 for the indicated times
    Figure Legend Snippet: MYH is active in HCT116 extracts and addresses G O /A but not A O /G or A O /C mispairs. A , nicking assay. The closed-circular homoduplex or the G O /A, A O /G, or A O /C substrates were incubated with recombinant, purified MYH-GST and APE1 for the indicated times

    Techniques Used: Incubation, Recombinant, Purification

    6) Product Images from "Oxidative modification of guanine in a potential Z-DNA-forming sequence of a gene promoter impacts gene expression"

    Article Title: Oxidative modification of guanine in a potential Z-DNA-forming sequence of a gene promoter impacts gene expression

    Journal: Chemical research in toxicology

    doi: 10.1021/acs.chemrestox.9b00041

    Efficiency of APE1-mediated cleavage of an F site in different structural contexts. ( A ) Sequences for the three contexts studied, and ( B ) the reaction yields at 5 and 30 min after initiation.
    Figure Legend Snippet: Efficiency of APE1-mediated cleavage of an F site in different structural contexts. ( A ) Sequences for the three contexts studied, and ( B ) the reaction yields at 5 and 30 min after initiation.

    Techniques Used:

    7) Product Images from "CUX1 stimulates APE1 enzymatic activity and increases the resistance of glioblastoma cells to the mono-alkylating agent temozolomide"

    Article Title: CUX1 stimulates APE1 enzymatic activity and increases the resistance of glioblastoma cells to the mono-alkylating agent temozolomide

    Journal: Neuro-Oncology

    doi: 10.1093/neuonc/nox178

    CUT domains stimulate APE1 endonuclease activity in vitro. (A) Schematic of CUX1 recombinant proteins used in APE1 endonuclease assays. (B, C) APE1 endonuclease assays were carried out using purified APE1, in the presence of purified CUX1 recombinant proteins or controls (BSA or homeobox B3) at 50 nM or as indicated, and a radiolabeled probe containing an abasic site produced in 2 different ways: as a tetrahydrofuran (B) or through removal of a uracil residue by UDG (C). (D) Schematic of the APE1 assay that utilizes a molecular beacon probe containing a tetrahydrofuran site. The APE1 assay was carried out with purified APE1 in the presence of nothing, 50 nM BSA, or purified CUX1 recombinant proteins (C2C3HD, C1C2, and C3HD), as indicated.
    Figure Legend Snippet: CUT domains stimulate APE1 endonuclease activity in vitro. (A) Schematic of CUX1 recombinant proteins used in APE1 endonuclease assays. (B, C) APE1 endonuclease assays were carried out using purified APE1, in the presence of purified CUX1 recombinant proteins or controls (BSA or homeobox B3) at 50 nM or as indicated, and a radiolabeled probe containing an abasic site produced in 2 different ways: as a tetrahydrofuran (B) or through removal of a uracil residue by UDG (C). (D) Schematic of the APE1 assay that utilizes a molecular beacon probe containing a tetrahydrofuran site. The APE1 assay was carried out with purified APE1 in the presence of nothing, 50 nM BSA, or purified CUX1 recombinant proteins (C2C3HD, C1C2, and C3HD), as indicated.

    Techniques Used: Activity Assay, In Vitro, Recombinant, Purification, Produced

    8) Product Images from "Deletion of Individual Ku Subunits in Mice Causes an NHEJ-Independent Phenotype Potentially by Altering Apurinic/Apyrimidinic Site Repair"

    Article Title: Deletion of Individual Ku Subunits in Mice Causes an NHEJ-Independent Phenotype Potentially by Altering Apurinic/Apyrimidinic Site Repair

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0086358

    Molecular beacon assay to measure APE1 activity. No Ku70 was compared to Ku70 added to substrate with or without APE1. Fluorescence: the excitation wavelength is 485 nm and the emission wavelength is 538 nm. Shown is the average of three experiments with error bars (standard deviation). (A) Ku70 1–609 (full-length Ku70) (B) Ku70 115–609 . (C) Ku70 1–115 . (D) Ku70 1–300 .
    Figure Legend Snippet: Molecular beacon assay to measure APE1 activity. No Ku70 was compared to Ku70 added to substrate with or without APE1. Fluorescence: the excitation wavelength is 485 nm and the emission wavelength is 538 nm. Shown is the average of three experiments with error bars (standard deviation). (A) Ku70 1–609 (full-length Ku70) (B) Ku70 115–609 . (C) Ku70 1–115 . (D) Ku70 1–300 .

    Techniques Used: Activity Assay, Fluorescence, Standard Deviation

    CRT0044876 (CRT) survival fraction (SF). All cells are deleted for p53 (even controls) to avoid early replicative senescence. Shown is the average of three experiments. (A) Cells deleted for Ku80 are hypersensitive to CRT. Expression of APE1 or mouse Ku80 rescued CRT hypersensitivity for Ku80-mutant cells. (B) Cells deleted for either Ku70 or Ku80 but not both were hypersensitive to CRT demonstrating independent function for the individual Ku subunits as opposed to the Ku heterodimer. (C) Cells deleted for Ku70 or Ku80 or both were hypersensitive to γ-radiation demonstrating the Ku heterodimer repaired damage as opposed to independent function for the individual proteins.
    Figure Legend Snippet: CRT0044876 (CRT) survival fraction (SF). All cells are deleted for p53 (even controls) to avoid early replicative senescence. Shown is the average of three experiments. (A) Cells deleted for Ku80 are hypersensitive to CRT. Expression of APE1 or mouse Ku80 rescued CRT hypersensitivity for Ku80-mutant cells. (B) Cells deleted for either Ku70 or Ku80 but not both were hypersensitive to CRT demonstrating independent function for the individual Ku subunits as opposed to the Ku heterodimer. (C) Cells deleted for Ku70 or Ku80 or both were hypersensitive to γ-radiation demonstrating the Ku heterodimer repaired damage as opposed to independent function for the individual proteins.

    Techniques Used: Expressing, Mutagenesis

    9) Product Images from "Widespread transcriptional gene inactivation initiated by a repair intermediate of 8-oxoguanine"

    Article Title: Widespread transcriptional gene inactivation initiated by a repair intermediate of 8-oxoguanine

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw473

    Inhibition of the EGFP gene expression by single apurinic site (AP). ( A ) Vectors used for site-specific insertion of synthetic oligonucleotides containing a single AP lesion (tetrahydrofuran) on the place of dG in the transcribed (TS) or the non-transcribed (NTS) strand of the EGFP gene. ( B ) Activity of APE1 toward plasmid substrates containing single synthetic AP lesion and the effects of the specified phosphorothioate linkages. ( C ) Fluorescence distribution plots of HeLa cells 24 h post-transfection with the expression constructs containing the indicated modifications in the specified DNA strand (TS or NTS), compared to the ‘G’ construct (overlaid blue line). Analogous plots for cells incubated in parallel with PARP inhibitors are shown in Supplementary Figure S5. ( D ) Mean relative EGFP expression of the specified constructs in the absence (DMSO) and in the presence of the indicated PARP inhibitors ( n = 3, ±SD).
    Figure Legend Snippet: Inhibition of the EGFP gene expression by single apurinic site (AP). ( A ) Vectors used for site-specific insertion of synthetic oligonucleotides containing a single AP lesion (tetrahydrofuran) on the place of dG in the transcribed (TS) or the non-transcribed (NTS) strand of the EGFP gene. ( B ) Activity of APE1 toward plasmid substrates containing single synthetic AP lesion and the effects of the specified phosphorothioate linkages. ( C ) Fluorescence distribution plots of HeLa cells 24 h post-transfection with the expression constructs containing the indicated modifications in the specified DNA strand (TS or NTS), compared to the ‘G’ construct (overlaid blue line). Analogous plots for cells incubated in parallel with PARP inhibitors are shown in Supplementary Figure S5. ( D ) Mean relative EGFP expression of the specified constructs in the absence (DMSO) and in the presence of the indicated PARP inhibitors ( n = 3, ±SD).

    Techniques Used: Inhibition, Expressing, Activity Assay, Plasmid Preparation, Fluorescence, Transfection, Construct, Incubation

    10) Product Images from "Development and evaluation of human AP endonuclease inhibitors in melanoma and glioma cell lines"

    Article Title: Development and evaluation of human AP endonuclease inhibitors in melanoma and glioma cell lines

    Journal: British Journal of Cancer

    doi: 10.1038/sj.bjc.6606058

    ( A ) AP endonuclease activity assays using 18-mer radiolabelled oligonucleotide substrates (see Materials and methods) were performed using HeLa whole-cell extracts (WCE). Compound 4 showed 93% inhibition of AP cleavage activity using WCE. ( B ) Compound 3, on the other hand did not inhibit WCE cleavage activity. ( C ) Testing AP site cleavage activity in wild-type and D148 polymorph. The figure shows that the activity was similar in both wild-type and the D148E polymorph. ( D ) Inhibitory activity of compound 4 against the D148E polymorphic variant of APE1 is shown here. D148E was more sensitive to inhibition by compound 4 compared with wild type.
    Figure Legend Snippet: ( A ) AP endonuclease activity assays using 18-mer radiolabelled oligonucleotide substrates (see Materials and methods) were performed using HeLa whole-cell extracts (WCE). Compound 4 showed 93% inhibition of AP cleavage activity using WCE. ( B ) Compound 3, on the other hand did not inhibit WCE cleavage activity. ( C ) Testing AP site cleavage activity in wild-type and D148 polymorph. The figure shows that the activity was similar in both wild-type and the D148E polymorph. ( D ) Inhibitory activity of compound 4 against the D148E polymorphic variant of APE1 is shown here. D148E was more sensitive to inhibition by compound 4 compared with wild type.

    Techniques Used: Activity Assay, Inhibition, Variant Assay

    Kinetics analysis. To evaluate potential mechanism of action of APE1 inhibitor, kinetic analysis was performed. Lineweaver–Burk plots and kinetic parameters determined from eight independent data points (note: error bars are in some cases too small to see) for compound 4 is shown here. The APE1 inhibitor was tested at three dose levels (5, 10 and 20 μ ) and oligonucleotide substrate was evaluated at three different concentrations (100, 200 and 500 n). The reaction was performed as described in methods. K M and k cat decreased at each inhibitor concentration (compared with no inhibitor) and the k cat /K M decreased at increasing inhibitor concentration. The data is consistent with uncompetitive inhibition.
    Figure Legend Snippet: Kinetics analysis. To evaluate potential mechanism of action of APE1 inhibitor, kinetic analysis was performed. Lineweaver–Burk plots and kinetic parameters determined from eight independent data points (note: error bars are in some cases too small to see) for compound 4 is shown here. The APE1 inhibitor was tested at three dose levels (5, 10 and 20 μ ) and oligonucleotide substrate was evaluated at three different concentrations (100, 200 and 500 n). The reaction was performed as described in methods. K M and k cat decreased at each inhibitor concentration (compared with no inhibitor) and the k cat /K M decreased at increasing inhibitor concentration. The data is consistent with uncompetitive inhibition.

    Techniques Used: Concentration Assay, Inhibition

    ( A ) Consensus score plot was constructed by plotting Gold Score ( x -axis) and Chem Score ( y -axis) for the 1679 virtual APE1 inhibitor candidates. The top ranking 25% of the compounds were shortlisted from the consensus plot and subjected to detailed biochemical analyses. ( B ) Primary screening. Fluorescence-based APE1 cleavage assay is shown here. If the DNA is cleaved at the abasic site at position 7 from the 5′ end by APE1, the 6-mer fluorescein-containing product will dissociate from its complement by thermal melting. As a result, the quenching effect of the 3′ dabcyl (which absorbs fluorescein fluorescence when in close proximity) is lost, and APE1 activity is measured indirectly as an increase in fluorescence signal. For detailed protocol see Materials and methods section. ( C ) APE1 inhibition by CRT0044876 is shown here. Control=no APE1 in reaction. ( D ) APE1 inhibition by compound 4 is shown here (IC 50 =11 μ ).
    Figure Legend Snippet: ( A ) Consensus score plot was constructed by plotting Gold Score ( x -axis) and Chem Score ( y -axis) for the 1679 virtual APE1 inhibitor candidates. The top ranking 25% of the compounds were shortlisted from the consensus plot and subjected to detailed biochemical analyses. ( B ) Primary screening. Fluorescence-based APE1 cleavage assay is shown here. If the DNA is cleaved at the abasic site at position 7 from the 5′ end by APE1, the 6-mer fluorescein-containing product will dissociate from its complement by thermal melting. As a result, the quenching effect of the 3′ dabcyl (which absorbs fluorescein fluorescence when in close proximity) is lost, and APE1 activity is measured indirectly as an increase in fluorescence signal. For detailed protocol see Materials and methods section. ( C ) APE1 inhibition by CRT0044876 is shown here. Control=no APE1 in reaction. ( D ) APE1 inhibition by compound 4 is shown here (IC 50 =11 μ ).

    Techniques Used: Construct, Fluorescence, Cleavage Assay, Activity Assay, Inhibition

    ( A ) APE1 expression in melanoma cell lines is shown here. ( B ) Inhibitor alone at 10 μ was relatively nontoxic to cells (as shown in Figure 7D ). We took the survival fraction as 100%. The percentage survival for those cells exposed to both inhibitor and temozolomide was plotted as a relative survival to cells exposed to the inhibitor alone. Potentiation of cytotoxicity of MMS by compound 4 (10 μ ) in SK-Mel30 cell line is shown here. ( C ) Potentiation of temozolomide by compound 4(10 μ ) in SK-Mel30 cell line is shown here. ( D ) Toxicity of compound 4 in HUVEC, SK-Mel30 and U89MG is shown here. Compound 4 was relatively nontoxic to HUVEC cells.
    Figure Legend Snippet: ( A ) APE1 expression in melanoma cell lines is shown here. ( B ) Inhibitor alone at 10 μ was relatively nontoxic to cells (as shown in Figure 7D ). We took the survival fraction as 100%. The percentage survival for those cells exposed to both inhibitor and temozolomide was plotted as a relative survival to cells exposed to the inhibitor alone. Potentiation of cytotoxicity of MMS by compound 4 (10 μ ) in SK-Mel30 cell line is shown here. ( C ) Potentiation of temozolomide by compound 4(10 μ ) in SK-Mel30 cell line is shown here. ( D ) Toxicity of compound 4 in HUVEC, SK-Mel30 and U89MG is shown here. Compound 4 was relatively nontoxic to HUVEC cells.

    Techniques Used: Expressing

    Secondary biochemical screening. ( A ) Fluorescence-based endonuclease IV cleavage assay is shown here. Compound 4 was tested at 100 μ and showed no inhibition of endonuclease IV. Control=no endonuclease IV in reaction ( B ). Compound 7 was tested at 100 μ and showed complete inhibition of endonuclease IV. Control=no endonuclease IV in reaction ( C ) Fluorescence queching assay did not reveal any quenching effect by compound 4. Control=no inhibitor in reaction ( D ). Radiolabelled assay showing inhibition of AP-site cleavage by APE1. Absence of 8-mer lower band indicates APE1 inhibition. See Material and methods for protcol details.
    Figure Legend Snippet: Secondary biochemical screening. ( A ) Fluorescence-based endonuclease IV cleavage assay is shown here. Compound 4 was tested at 100 μ and showed no inhibition of endonuclease IV. Control=no endonuclease IV in reaction ( B ). Compound 7 was tested at 100 μ and showed complete inhibition of endonuclease IV. Control=no endonuclease IV in reaction ( C ) Fluorescence queching assay did not reveal any quenching effect by compound 4. Control=no inhibitor in reaction ( D ). Radiolabelled assay showing inhibition of AP-site cleavage by APE1. Absence of 8-mer lower band indicates APE1 inhibition. See Material and methods for protcol details.

    Techniques Used: Fluorescence, Cleavage Assay, Inhibition

    Molecular Modelling. ( A ) The DNA repair domain active site was localised on the basis of the previously reported 10 critical amino acid residues that are essential for the AP endonuclease activity of APE1 (D70, D90, E96, Y171, D210, N212, D219, D283, D308 and H309, see text for details). Visual Molecular Dynamics1.8.7 was used to visualise the crystal structure of APE1. The molecular surface in the region of the V-shaped active site cleft is shown here. ( B ) Sybyl8.0 was used to build inhibitor templates. Chemical structures and docked poses of the four prototypical ligands onto APE1 active site are shown here.
    Figure Legend Snippet: Molecular Modelling. ( A ) The DNA repair domain active site was localised on the basis of the previously reported 10 critical amino acid residues that are essential for the AP endonuclease activity of APE1 (D70, D90, E96, Y171, D210, N212, D219, D283, D308 and H309, see text for details). Visual Molecular Dynamics1.8.7 was used to visualise the crystal structure of APE1. The molecular surface in the region of the V-shaped active site cleft is shown here. ( B ) Sybyl8.0 was used to build inhibitor templates. Chemical structures and docked poses of the four prototypical ligands onto APE1 active site are shown here.

    Techniques Used: Activity Assay

    ( A ) APE1 expression in glioma cell lines is shown here. ( B ) (1 and 2). Aldehyde reactive probe assay. U89MG cells were pretreated with 10 μ of compound 4 alone or MMS (600 μ ) or a combination of compound 4 and MMS. Genomic DNA extracted at 90 min for AP-site quatification. The combination treatment led to increased AP site content in the genomic DNA. ( C ) Inhibitor alone at 10 μ was relatively nontoxic to cells (as shown in Figure 7D ). We took the survival fraction as 100%. The percentage survival for those cells exposed to both inhibitor and temozolomide was plotted as a relative survival to cells exposed to the inhibitor alone. Potentiation of cytotoxicity of MMS by compound 4 (10 μ ) in U89 MG cell line is shown here. ( D ) Potentiation of temozolomide by compound 4 (10 μ ) in U89MG cell line is shown here.
    Figure Legend Snippet: ( A ) APE1 expression in glioma cell lines is shown here. ( B ) (1 and 2). Aldehyde reactive probe assay. U89MG cells were pretreated with 10 μ of compound 4 alone or MMS (600 μ ) or a combination of compound 4 and MMS. Genomic DNA extracted at 90 min for AP-site quatification. The combination treatment led to increased AP site content in the genomic DNA. ( C ) Inhibitor alone at 10 μ was relatively nontoxic to cells (as shown in Figure 7D ). We took the survival fraction as 100%. The percentage survival for those cells exposed to both inhibitor and temozolomide was plotted as a relative survival to cells exposed to the inhibitor alone. Potentiation of cytotoxicity of MMS by compound 4 (10 μ ) in U89 MG cell line is shown here. ( D ) Potentiation of temozolomide by compound 4 (10 μ ) in U89MG cell line is shown here.

    Techniques Used: Expressing

    11) Product Images from "Development and evaluation of human AP endonuclease inhibitors in melanoma and glioma cell lines"

    Article Title: Development and evaluation of human AP endonuclease inhibitors in melanoma and glioma cell lines

    Journal: British Journal of Cancer

    doi: 10.1038/sj.bjc.6606058

    ( A ) AP endonuclease activity assays using 18-mer radiolabelled oligonucleotide substrates (see Materials and methods) were performed using HeLa whole-cell extracts (WCE). Compound 4 showed 93% inhibition of AP cleavage activity using WCE. ( B ) Compound 3, on the other hand did not inhibit WCE cleavage activity. ( C ) Testing AP site cleavage activity in wild-type and D148 polymorph. The figure shows that the activity was similar in both wild-type and the D148E polymorph. ( D ) Inhibitory activity of compound 4 against the D148E polymorphic variant of APE1 is shown here. D148E was more sensitive to inhibition by compound 4 compared with wild type.
    Figure Legend Snippet: ( A ) AP endonuclease activity assays using 18-mer radiolabelled oligonucleotide substrates (see Materials and methods) were performed using HeLa whole-cell extracts (WCE). Compound 4 showed 93% inhibition of AP cleavage activity using WCE. ( B ) Compound 3, on the other hand did not inhibit WCE cleavage activity. ( C ) Testing AP site cleavage activity in wild-type and D148 polymorph. The figure shows that the activity was similar in both wild-type and the D148E polymorph. ( D ) Inhibitory activity of compound 4 against the D148E polymorphic variant of APE1 is shown here. D148E was more sensitive to inhibition by compound 4 compared with wild type.

    Techniques Used: Activity Assay, Inhibition, Variant Assay

    Kinetics analysis. To evaluate potential mechanism of action of APE1 inhibitor, kinetic analysis was performed. Lineweaver–Burk plots and kinetic parameters determined from eight independent data points (note: error bars are in some cases too small to see) for compound 4 is shown here. The APE1 inhibitor was tested at three dose levels (5, 10 and 20 μ ) and oligonucleotide substrate was evaluated at three different concentrations (100, 200 and 500 n). The reaction was performed as described in methods. K M and k cat decreased at each inhibitor concentration (compared with no inhibitor) and the k cat /K M decreased at increasing inhibitor concentration. The data is consistent with uncompetitive inhibition.
    Figure Legend Snippet: Kinetics analysis. To evaluate potential mechanism of action of APE1 inhibitor, kinetic analysis was performed. Lineweaver–Burk plots and kinetic parameters determined from eight independent data points (note: error bars are in some cases too small to see) for compound 4 is shown here. The APE1 inhibitor was tested at three dose levels (5, 10 and 20 μ ) and oligonucleotide substrate was evaluated at three different concentrations (100, 200 and 500 n). The reaction was performed as described in methods. K M and k cat decreased at each inhibitor concentration (compared with no inhibitor) and the k cat /K M decreased at increasing inhibitor concentration. The data is consistent with uncompetitive inhibition.

    Techniques Used: Concentration Assay, Inhibition

    ( A ) Consensus score plot was constructed by plotting Gold Score ( x -axis) and Chem Score ( y -axis) for the 1679 virtual APE1 inhibitor candidates. The top ranking 25% of the compounds were shortlisted from the consensus plot and subjected to detailed biochemical analyses. ( B ) Primary screening. Fluorescence-based APE1 cleavage assay is shown here. If the DNA is cleaved at the abasic site at position 7 from the 5′ end by APE1, the 6-mer fluorescein-containing product will dissociate from its complement by thermal melting. As a result, the quenching effect of the 3′ dabcyl (which absorbs fluorescein fluorescence when in close proximity) is lost, and APE1 activity is measured indirectly as an increase in fluorescence signal. For detailed protocol see Materials and methods section. ( C ) APE1 inhibition by CRT0044876 is shown here. Control=no APE1 in reaction. ( D ) APE1 inhibition by compound 4 is shown here (IC 50 =11 μ ).
    Figure Legend Snippet: ( A ) Consensus score plot was constructed by plotting Gold Score ( x -axis) and Chem Score ( y -axis) for the 1679 virtual APE1 inhibitor candidates. The top ranking 25% of the compounds were shortlisted from the consensus plot and subjected to detailed biochemical analyses. ( B ) Primary screening. Fluorescence-based APE1 cleavage assay is shown here. If the DNA is cleaved at the abasic site at position 7 from the 5′ end by APE1, the 6-mer fluorescein-containing product will dissociate from its complement by thermal melting. As a result, the quenching effect of the 3′ dabcyl (which absorbs fluorescein fluorescence when in close proximity) is lost, and APE1 activity is measured indirectly as an increase in fluorescence signal. For detailed protocol see Materials and methods section. ( C ) APE1 inhibition by CRT0044876 is shown here. Control=no APE1 in reaction. ( D ) APE1 inhibition by compound 4 is shown here (IC 50 =11 μ ).

    Techniques Used: Construct, Fluorescence, Cleavage Assay, Activity Assay, Inhibition

    ( A ) APE1 expression in melanoma cell lines is shown here. ( B ) Inhibitor alone at 10 μ was relatively nontoxic to cells (as shown in Figure 7D ). We took the survival fraction as 100%. The percentage survival for those cells exposed to both inhibitor and temozolomide was plotted as a relative survival to cells exposed to the inhibitor alone. Potentiation of cytotoxicity of MMS by compound 4 (10 μ ) in SK-Mel30 cell line is shown here. ( C ) Potentiation of temozolomide by compound 4(10 μ ) in SK-Mel30 cell line is shown here. ( D ) Toxicity of compound 4 in HUVEC, SK-Mel30 and U89MG is shown here. Compound 4 was relatively nontoxic to HUVEC cells.
    Figure Legend Snippet: ( A ) APE1 expression in melanoma cell lines is shown here. ( B ) Inhibitor alone at 10 μ was relatively nontoxic to cells (as shown in Figure 7D ). We took the survival fraction as 100%. The percentage survival for those cells exposed to both inhibitor and temozolomide was plotted as a relative survival to cells exposed to the inhibitor alone. Potentiation of cytotoxicity of MMS by compound 4 (10 μ ) in SK-Mel30 cell line is shown here. ( C ) Potentiation of temozolomide by compound 4(10 μ ) in SK-Mel30 cell line is shown here. ( D ) Toxicity of compound 4 in HUVEC, SK-Mel30 and U89MG is shown here. Compound 4 was relatively nontoxic to HUVEC cells.

    Techniques Used: Expressing

    Secondary biochemical screening. ( A ) Fluorescence-based endonuclease IV cleavage assay is shown here. Compound 4 was tested at 100 μ and showed no inhibition of endonuclease IV. Control=no endonuclease IV in reaction ( B ). Compound 7 was tested at 100 μ and showed complete inhibition of endonuclease IV. Control=no endonuclease IV in reaction ( C ) Fluorescence queching assay did not reveal any quenching effect by compound 4. Control=no inhibitor in reaction ( D ). Radiolabelled assay showing inhibition of AP-site cleavage by APE1. Absence of 8-mer lower band indicates APE1 inhibition. See Material and methods for protcol details.
    Figure Legend Snippet: Secondary biochemical screening. ( A ) Fluorescence-based endonuclease IV cleavage assay is shown here. Compound 4 was tested at 100 μ and showed no inhibition of endonuclease IV. Control=no endonuclease IV in reaction ( B ). Compound 7 was tested at 100 μ and showed complete inhibition of endonuclease IV. Control=no endonuclease IV in reaction ( C ) Fluorescence queching assay did not reveal any quenching effect by compound 4. Control=no inhibitor in reaction ( D ). Radiolabelled assay showing inhibition of AP-site cleavage by APE1. Absence of 8-mer lower band indicates APE1 inhibition. See Material and methods for protcol details.

    Techniques Used: Fluorescence, Cleavage Assay, Inhibition

    Molecular Modelling. ( A ) The DNA repair domain active site was localised on the basis of the previously reported 10 critical amino acid residues that are essential for the AP endonuclease activity of APE1 (D70, D90, E96, Y171, D210, N212, D219, D283, D308 and H309, see text for details). Visual Molecular Dynamics1.8.7 was used to visualise the crystal structure of APE1. The molecular surface in the region of the V-shaped active site cleft is shown here. ( B ) Sybyl8.0 was used to build inhibitor templates. Chemical structures and docked poses of the four prototypical ligands onto APE1 active site are shown here.
    Figure Legend Snippet: Molecular Modelling. ( A ) The DNA repair domain active site was localised on the basis of the previously reported 10 critical amino acid residues that are essential for the AP endonuclease activity of APE1 (D70, D90, E96, Y171, D210, N212, D219, D283, D308 and H309, see text for details). Visual Molecular Dynamics1.8.7 was used to visualise the crystal structure of APE1. The molecular surface in the region of the V-shaped active site cleft is shown here. ( B ) Sybyl8.0 was used to build inhibitor templates. Chemical structures and docked poses of the four prototypical ligands onto APE1 active site are shown here.

    Techniques Used: Activity Assay

    ( A ) APE1 expression in glioma cell lines is shown here. ( B ) (1 and 2). Aldehyde reactive probe assay. U89MG cells were pretreated with 10 μ of compound 4 alone or MMS (600 μ ) or a combination of compound 4 and MMS. Genomic DNA extracted at 90 min for AP-site quatification. The combination treatment led to increased AP site content in the genomic DNA. ( C ) Inhibitor alone at 10 μ was relatively nontoxic to cells (as shown in Figure 7D ). We took the survival fraction as 100%. The percentage survival for those cells exposed to both inhibitor and temozolomide was plotted as a relative survival to cells exposed to the inhibitor alone. Potentiation of cytotoxicity of MMS by compound 4 (10 μ ) in U89 MG cell line is shown here. ( D ) Potentiation of temozolomide by compound 4 (10 μ ) in U89MG cell line is shown here.
    Figure Legend Snippet: ( A ) APE1 expression in glioma cell lines is shown here. ( B ) (1 and 2). Aldehyde reactive probe assay. U89MG cells were pretreated with 10 μ of compound 4 alone or MMS (600 μ ) or a combination of compound 4 and MMS. Genomic DNA extracted at 90 min for AP-site quatification. The combination treatment led to increased AP site content in the genomic DNA. ( C ) Inhibitor alone at 10 μ was relatively nontoxic to cells (as shown in Figure 7D ). We took the survival fraction as 100%. The percentage survival for those cells exposed to both inhibitor and temozolomide was plotted as a relative survival to cells exposed to the inhibitor alone. Potentiation of cytotoxicity of MMS by compound 4 (10 μ ) in U89 MG cell line is shown here. ( D ) Potentiation of temozolomide by compound 4 (10 μ ) in U89MG cell line is shown here.

    Techniques Used: Expressing

    12) Product Images from "Development and evaluation of human AP endonuclease inhibitors in melanoma and glioma cell lines"

    Article Title: Development and evaluation of human AP endonuclease inhibitors in melanoma and glioma cell lines

    Journal: British Journal of Cancer

    doi: 10.1038/sj.bjc.6606058

    ( A ) AP endonuclease activity assays using 18-mer radiolabelled oligonucleotide substrates (see Materials and methods) were performed using HeLa whole-cell extracts (WCE). Compound 4 showed 93% inhibition of AP cleavage activity using WCE. ( B ) Compound 3, on the other hand did not inhibit WCE cleavage activity. ( C ) Testing AP site cleavage activity in wild-type and D148 polymorph. The figure shows that the activity was similar in both wild-type and the D148E polymorph. ( D ) Inhibitory activity of compound 4 against the D148E polymorphic variant of APE1 is shown here. D148E was more sensitive to inhibition by compound 4 compared with wild type.
    Figure Legend Snippet: ( A ) AP endonuclease activity assays using 18-mer radiolabelled oligonucleotide substrates (see Materials and methods) were performed using HeLa whole-cell extracts (WCE). Compound 4 showed 93% inhibition of AP cleavage activity using WCE. ( B ) Compound 3, on the other hand did not inhibit WCE cleavage activity. ( C ) Testing AP site cleavage activity in wild-type and D148 polymorph. The figure shows that the activity was similar in both wild-type and the D148E polymorph. ( D ) Inhibitory activity of compound 4 against the D148E polymorphic variant of APE1 is shown here. D148E was more sensitive to inhibition by compound 4 compared with wild type.

    Techniques Used: Activity Assay, Inhibition, Variant Assay

    Kinetics analysis. To evaluate potential mechanism of action of APE1 inhibitor, kinetic analysis was performed. Lineweaver–Burk plots and kinetic parameters determined from eight independent data points (note: error bars are in some cases too small to see) for compound 4 is shown here. The APE1 inhibitor was tested at three dose levels (5, 10 and 20 μ ) and oligonucleotide substrate was evaluated at three different concentrations (100, 200 and 500 n). The reaction was performed as described in methods. K M and k cat decreased at each inhibitor concentration (compared with no inhibitor) and the k cat /K M decreased at increasing inhibitor concentration. The data is consistent with uncompetitive inhibition.
    Figure Legend Snippet: Kinetics analysis. To evaluate potential mechanism of action of APE1 inhibitor, kinetic analysis was performed. Lineweaver–Burk plots and kinetic parameters determined from eight independent data points (note: error bars are in some cases too small to see) for compound 4 is shown here. The APE1 inhibitor was tested at three dose levels (5, 10 and 20 μ ) and oligonucleotide substrate was evaluated at three different concentrations (100, 200 and 500 n). The reaction was performed as described in methods. K M and k cat decreased at each inhibitor concentration (compared with no inhibitor) and the k cat /K M decreased at increasing inhibitor concentration. The data is consistent with uncompetitive inhibition.

    Techniques Used: Concentration Assay, Inhibition

    ( A ) Consensus score plot was constructed by plotting Gold Score ( x -axis) and Chem Score ( y -axis) for the 1679 virtual APE1 inhibitor candidates. The top ranking 25% of the compounds were shortlisted from the consensus plot and subjected to detailed biochemical analyses. ( B ) Primary screening. Fluorescence-based APE1 cleavage assay is shown here. If the DNA is cleaved at the abasic site at position 7 from the 5′ end by APE1, the 6-mer fluorescein-containing product will dissociate from its complement by thermal melting. As a result, the quenching effect of the 3′ dabcyl (which absorbs fluorescein fluorescence when in close proximity) is lost, and APE1 activity is measured indirectly as an increase in fluorescence signal. For detailed protocol see Materials and methods section. ( C ) APE1 inhibition by CRT0044876 is shown here. Control=no APE1 in reaction. ( D ) APE1 inhibition by compound 4 is shown here (IC 50 =11 μ ).
    Figure Legend Snippet: ( A ) Consensus score plot was constructed by plotting Gold Score ( x -axis) and Chem Score ( y -axis) for the 1679 virtual APE1 inhibitor candidates. The top ranking 25% of the compounds were shortlisted from the consensus plot and subjected to detailed biochemical analyses. ( B ) Primary screening. Fluorescence-based APE1 cleavage assay is shown here. If the DNA is cleaved at the abasic site at position 7 from the 5′ end by APE1, the 6-mer fluorescein-containing product will dissociate from its complement by thermal melting. As a result, the quenching effect of the 3′ dabcyl (which absorbs fluorescein fluorescence when in close proximity) is lost, and APE1 activity is measured indirectly as an increase in fluorescence signal. For detailed protocol see Materials and methods section. ( C ) APE1 inhibition by CRT0044876 is shown here. Control=no APE1 in reaction. ( D ) APE1 inhibition by compound 4 is shown here (IC 50 =11 μ ).

    Techniques Used: Construct, Fluorescence, Cleavage Assay, Activity Assay, Inhibition

    ( A ) APE1 expression in melanoma cell lines is shown here. ( B ) Inhibitor alone at 10 μ was relatively nontoxic to cells (as shown in Figure 7D ). We took the survival fraction as 100%. The percentage survival for those cells exposed to both inhibitor and temozolomide was plotted as a relative survival to cells exposed to the inhibitor alone. Potentiation of cytotoxicity of MMS by compound 4 (10 μ ) in SK-Mel30 cell line is shown here. ( C ) Potentiation of temozolomide by compound 4(10 μ ) in SK-Mel30 cell line is shown here. ( D ) Toxicity of compound 4 in HUVEC, SK-Mel30 and U89MG is shown here. Compound 4 was relatively nontoxic to HUVEC cells.
    Figure Legend Snippet: ( A ) APE1 expression in melanoma cell lines is shown here. ( B ) Inhibitor alone at 10 μ was relatively nontoxic to cells (as shown in Figure 7D ). We took the survival fraction as 100%. The percentage survival for those cells exposed to both inhibitor and temozolomide was plotted as a relative survival to cells exposed to the inhibitor alone. Potentiation of cytotoxicity of MMS by compound 4 (10 μ ) in SK-Mel30 cell line is shown here. ( C ) Potentiation of temozolomide by compound 4(10 μ ) in SK-Mel30 cell line is shown here. ( D ) Toxicity of compound 4 in HUVEC, SK-Mel30 and U89MG is shown here. Compound 4 was relatively nontoxic to HUVEC cells.

    Techniques Used: Expressing

    Secondary biochemical screening. ( A ) Fluorescence-based endonuclease IV cleavage assay is shown here. Compound 4 was tested at 100 μ and showed no inhibition of endonuclease IV. Control=no endonuclease IV in reaction ( B ). Compound 7 was tested at 100 μ and showed complete inhibition of endonuclease IV. Control=no endonuclease IV in reaction ( C ) Fluorescence queching assay did not reveal any quenching effect by compound 4. Control=no inhibitor in reaction ( D ). Radiolabelled assay showing inhibition of AP-site cleavage by APE1. Absence of 8-mer lower band indicates APE1 inhibition. See Material and methods for protcol details.
    Figure Legend Snippet: Secondary biochemical screening. ( A ) Fluorescence-based endonuclease IV cleavage assay is shown here. Compound 4 was tested at 100 μ and showed no inhibition of endonuclease IV. Control=no endonuclease IV in reaction ( B ). Compound 7 was tested at 100 μ and showed complete inhibition of endonuclease IV. Control=no endonuclease IV in reaction ( C ) Fluorescence queching assay did not reveal any quenching effect by compound 4. Control=no inhibitor in reaction ( D ). Radiolabelled assay showing inhibition of AP-site cleavage by APE1. Absence of 8-mer lower band indicates APE1 inhibition. See Material and methods for protcol details.

    Techniques Used: Fluorescence, Cleavage Assay, Inhibition

    Molecular Modelling. ( A ) The DNA repair domain active site was localised on the basis of the previously reported 10 critical amino acid residues that are essential for the AP endonuclease activity of APE1 (D70, D90, E96, Y171, D210, N212, D219, D283, D308 and H309, see text for details). Visual Molecular Dynamics1.8.7 was used to visualise the crystal structure of APE1. The molecular surface in the region of the V-shaped active site cleft is shown here. ( B ) Sybyl8.0 was used to build inhibitor templates. Chemical structures and docked poses of the four prototypical ligands onto APE1 active site are shown here.
    Figure Legend Snippet: Molecular Modelling. ( A ) The DNA repair domain active site was localised on the basis of the previously reported 10 critical amino acid residues that are essential for the AP endonuclease activity of APE1 (D70, D90, E96, Y171, D210, N212, D219, D283, D308 and H309, see text for details). Visual Molecular Dynamics1.8.7 was used to visualise the crystal structure of APE1. The molecular surface in the region of the V-shaped active site cleft is shown here. ( B ) Sybyl8.0 was used to build inhibitor templates. Chemical structures and docked poses of the four prototypical ligands onto APE1 active site are shown here.

    Techniques Used: Activity Assay

    ( A ) APE1 expression in glioma cell lines is shown here. ( B ) (1 and 2). Aldehyde reactive probe assay. U89MG cells were pretreated with 10 μ of compound 4 alone or MMS (600 μ ) or a combination of compound 4 and MMS. Genomic DNA extracted at 90 min for AP-site quatification. The combination treatment led to increased AP site content in the genomic DNA. ( C ) Inhibitor alone at 10 μ was relatively nontoxic to cells (as shown in Figure 7D ). We took the survival fraction as 100%. The percentage survival for those cells exposed to both inhibitor and temozolomide was plotted as a relative survival to cells exposed to the inhibitor alone. Potentiation of cytotoxicity of MMS by compound 4 (10 μ ) in U89 MG cell line is shown here. ( D ) Potentiation of temozolomide by compound 4 (10 μ ) in U89MG cell line is shown here.
    Figure Legend Snippet: ( A ) APE1 expression in glioma cell lines is shown here. ( B ) (1 and 2). Aldehyde reactive probe assay. U89MG cells were pretreated with 10 μ of compound 4 alone or MMS (600 μ ) or a combination of compound 4 and MMS. Genomic DNA extracted at 90 min for AP-site quatification. The combination treatment led to increased AP site content in the genomic DNA. ( C ) Inhibitor alone at 10 μ was relatively nontoxic to cells (as shown in Figure 7D ). We took the survival fraction as 100%. The percentage survival for those cells exposed to both inhibitor and temozolomide was plotted as a relative survival to cells exposed to the inhibitor alone. Potentiation of cytotoxicity of MMS by compound 4 (10 μ ) in U89 MG cell line is shown here. ( D ) Potentiation of temozolomide by compound 4 (10 μ ) in U89MG cell line is shown here.

    Techniques Used: Expressing

    13) Product Images from "Development and evaluation of human AP endonuclease inhibitors in melanoma and glioma cell lines"

    Article Title: Development and evaluation of human AP endonuclease inhibitors in melanoma and glioma cell lines

    Journal: British Journal of Cancer

    doi: 10.1038/sj.bjc.6606058

    ( A ) AP endonuclease activity assays using 18-mer radiolabelled oligonucleotide substrates (see Materials and methods) were performed using HeLa whole-cell extracts (WCE). Compound 4 showed 93% inhibition of AP cleavage activity using WCE. ( B ) Compound 3, on the other hand did not inhibit WCE cleavage activity. ( C ) Testing AP site cleavage activity in wild-type and D148 polymorph. The figure shows that the activity was similar in both wild-type and the D148E polymorph. ( D ) Inhibitory activity of compound 4 against the D148E polymorphic variant of APE1 is shown here. D148E was more sensitive to inhibition by compound 4 compared with wild type.
    Figure Legend Snippet: ( A ) AP endonuclease activity assays using 18-mer radiolabelled oligonucleotide substrates (see Materials and methods) were performed using HeLa whole-cell extracts (WCE). Compound 4 showed 93% inhibition of AP cleavage activity using WCE. ( B ) Compound 3, on the other hand did not inhibit WCE cleavage activity. ( C ) Testing AP site cleavage activity in wild-type and D148 polymorph. The figure shows that the activity was similar in both wild-type and the D148E polymorph. ( D ) Inhibitory activity of compound 4 against the D148E polymorphic variant of APE1 is shown here. D148E was more sensitive to inhibition by compound 4 compared with wild type.

    Techniques Used: Activity Assay, Inhibition, Variant Assay

    Kinetics analysis. To evaluate potential mechanism of action of APE1 inhibitor, kinetic analysis was performed. Lineweaver–Burk plots and kinetic parameters determined from eight independent data points (note: error bars are in some cases too small to see) for compound 4 is shown here. The APE1 inhibitor was tested at three dose levels (5, 10 and 20 μ ) and oligonucleotide substrate was evaluated at three different concentrations (100, 200 and 500 n). The reaction was performed as described in methods. K M and k cat decreased at each inhibitor concentration (compared with no inhibitor) and the k cat /K M decreased at increasing inhibitor concentration. The data is consistent with uncompetitive inhibition.
    Figure Legend Snippet: Kinetics analysis. To evaluate potential mechanism of action of APE1 inhibitor, kinetic analysis was performed. Lineweaver–Burk plots and kinetic parameters determined from eight independent data points (note: error bars are in some cases too small to see) for compound 4 is shown here. The APE1 inhibitor was tested at three dose levels (5, 10 and 20 μ ) and oligonucleotide substrate was evaluated at three different concentrations (100, 200 and 500 n). The reaction was performed as described in methods. K M and k cat decreased at each inhibitor concentration (compared with no inhibitor) and the k cat /K M decreased at increasing inhibitor concentration. The data is consistent with uncompetitive inhibition.

    Techniques Used: Concentration Assay, Inhibition

    ( A ) Consensus score plot was constructed by plotting Gold Score ( x -axis) and Chem Score ( y -axis) for the 1679 virtual APE1 inhibitor candidates. The top ranking 25% of the compounds were shortlisted from the consensus plot and subjected to detailed biochemical analyses. ( B ) Primary screening. Fluorescence-based APE1 cleavage assay is shown here. If the DNA is cleaved at the abasic site at position 7 from the 5′ end by APE1, the 6-mer fluorescein-containing product will dissociate from its complement by thermal melting. As a result, the quenching effect of the 3′ dabcyl (which absorbs fluorescein fluorescence when in close proximity) is lost, and APE1 activity is measured indirectly as an increase in fluorescence signal. For detailed protocol see Materials and methods section. ( C ) APE1 inhibition by CRT0044876 is shown here. Control=no APE1 in reaction. ( D ) APE1 inhibition by compound 4 is shown here (IC 50 =11 μ ).
    Figure Legend Snippet: ( A ) Consensus score plot was constructed by plotting Gold Score ( x -axis) and Chem Score ( y -axis) for the 1679 virtual APE1 inhibitor candidates. The top ranking 25% of the compounds were shortlisted from the consensus plot and subjected to detailed biochemical analyses. ( B ) Primary screening. Fluorescence-based APE1 cleavage assay is shown here. If the DNA is cleaved at the abasic site at position 7 from the 5′ end by APE1, the 6-mer fluorescein-containing product will dissociate from its complement by thermal melting. As a result, the quenching effect of the 3′ dabcyl (which absorbs fluorescein fluorescence when in close proximity) is lost, and APE1 activity is measured indirectly as an increase in fluorescence signal. For detailed protocol see Materials and methods section. ( C ) APE1 inhibition by CRT0044876 is shown here. Control=no APE1 in reaction. ( D ) APE1 inhibition by compound 4 is shown here (IC 50 =11 μ ).

    Techniques Used: Construct, Fluorescence, Cleavage Assay, Activity Assay, Inhibition

    ( A ) APE1 expression in melanoma cell lines is shown here. ( B ) Inhibitor alone at 10 μ was relatively nontoxic to cells (as shown in Figure 7D ). We took the survival fraction as 100%. The percentage survival for those cells exposed to both inhibitor and temozolomide was plotted as a relative survival to cells exposed to the inhibitor alone. Potentiation of cytotoxicity of MMS by compound 4 (10 μ ) in SK-Mel30 cell line is shown here. ( C ) Potentiation of temozolomide by compound 4(10 μ ) in SK-Mel30 cell line is shown here. ( D ) Toxicity of compound 4 in HUVEC, SK-Mel30 and U89MG is shown here. Compound 4 was relatively nontoxic to HUVEC cells.
    Figure Legend Snippet: ( A ) APE1 expression in melanoma cell lines is shown here. ( B ) Inhibitor alone at 10 μ was relatively nontoxic to cells (as shown in Figure 7D ). We took the survival fraction as 100%. The percentage survival for those cells exposed to both inhibitor and temozolomide was plotted as a relative survival to cells exposed to the inhibitor alone. Potentiation of cytotoxicity of MMS by compound 4 (10 μ ) in SK-Mel30 cell line is shown here. ( C ) Potentiation of temozolomide by compound 4(10 μ ) in SK-Mel30 cell line is shown here. ( D ) Toxicity of compound 4 in HUVEC, SK-Mel30 and U89MG is shown here. Compound 4 was relatively nontoxic to HUVEC cells.

    Techniques Used: Expressing

    Secondary biochemical screening. ( A ) Fluorescence-based endonuclease IV cleavage assay is shown here. Compound 4 was tested at 100 μ and showed no inhibition of endonuclease IV. Control=no endonuclease IV in reaction ( B ). Compound 7 was tested at 100 μ and showed complete inhibition of endonuclease IV. Control=no endonuclease IV in reaction ( C ) Fluorescence queching assay did not reveal any quenching effect by compound 4. Control=no inhibitor in reaction ( D ). Radiolabelled assay showing inhibition of AP-site cleavage by APE1. Absence of 8-mer lower band indicates APE1 inhibition. See Material and methods for protcol details.
    Figure Legend Snippet: Secondary biochemical screening. ( A ) Fluorescence-based endonuclease IV cleavage assay is shown here. Compound 4 was tested at 100 μ and showed no inhibition of endonuclease IV. Control=no endonuclease IV in reaction ( B ). Compound 7 was tested at 100 μ and showed complete inhibition of endonuclease IV. Control=no endonuclease IV in reaction ( C ) Fluorescence queching assay did not reveal any quenching effect by compound 4. Control=no inhibitor in reaction ( D ). Radiolabelled assay showing inhibition of AP-site cleavage by APE1. Absence of 8-mer lower band indicates APE1 inhibition. See Material and methods for protcol details.

    Techniques Used: Fluorescence, Cleavage Assay, Inhibition

    Molecular Modelling. ( A ) The DNA repair domain active site was localised on the basis of the previously reported 10 critical amino acid residues that are essential for the AP endonuclease activity of APE1 (D70, D90, E96, Y171, D210, N212, D219, D283, D308 and H309, see text for details). Visual Molecular Dynamics1.8.7 was used to visualise the crystal structure of APE1. The molecular surface in the region of the V-shaped active site cleft is shown here. ( B ) Sybyl8.0 was used to build inhibitor templates. Chemical structures and docked poses of the four prototypical ligands onto APE1 active site are shown here.
    Figure Legend Snippet: Molecular Modelling. ( A ) The DNA repair domain active site was localised on the basis of the previously reported 10 critical amino acid residues that are essential for the AP endonuclease activity of APE1 (D70, D90, E96, Y171, D210, N212, D219, D283, D308 and H309, see text for details). Visual Molecular Dynamics1.8.7 was used to visualise the crystal structure of APE1. The molecular surface in the region of the V-shaped active site cleft is shown here. ( B ) Sybyl8.0 was used to build inhibitor templates. Chemical structures and docked poses of the four prototypical ligands onto APE1 active site are shown here.

    Techniques Used: Activity Assay

    ( A ) APE1 expression in glioma cell lines is shown here. ( B ) (1 and 2). Aldehyde reactive probe assay. U89MG cells were pretreated with 10 μ of compound 4 alone or MMS (600 μ ) or a combination of compound 4 and MMS. Genomic DNA extracted at 90 min for AP-site quatification. The combination treatment led to increased AP site content in the genomic DNA. ( C ) Inhibitor alone at 10 μ was relatively nontoxic to cells (as shown in Figure 7D ). We took the survival fraction as 100%. The percentage survival for those cells exposed to both inhibitor and temozolomide was plotted as a relative survival to cells exposed to the inhibitor alone. Potentiation of cytotoxicity of MMS by compound 4 (10 μ ) in U89 MG cell line is shown here. ( D ) Potentiation of temozolomide by compound 4 (10 μ ) in U89MG cell line is shown here.
    Figure Legend Snippet: ( A ) APE1 expression in glioma cell lines is shown here. ( B ) (1 and 2). Aldehyde reactive probe assay. U89MG cells were pretreated with 10 μ of compound 4 alone or MMS (600 μ ) or a combination of compound 4 and MMS. Genomic DNA extracted at 90 min for AP-site quatification. The combination treatment led to increased AP site content in the genomic DNA. ( C ) Inhibitor alone at 10 μ was relatively nontoxic to cells (as shown in Figure 7D ). We took the survival fraction as 100%. The percentage survival for those cells exposed to both inhibitor and temozolomide was plotted as a relative survival to cells exposed to the inhibitor alone. Potentiation of cytotoxicity of MMS by compound 4 (10 μ ) in U89 MG cell line is shown here. ( D ) Potentiation of temozolomide by compound 4 (10 μ ) in U89MG cell line is shown here.

    Techniques Used: Expressing

    14) Product Images from "Development and evaluation of human AP endonuclease inhibitors in melanoma and glioma cell lines"

    Article Title: Development and evaluation of human AP endonuclease inhibitors in melanoma and glioma cell lines

    Journal: British Journal of Cancer

    doi: 10.1038/sj.bjc.6606058

    ( A ) AP endonuclease activity assays using 18-mer radiolabelled oligonucleotide substrates (see Materials and methods) were performed using HeLa whole-cell extracts (WCE). Compound 4 showed 93% inhibition of AP cleavage activity using WCE. ( B ) Compound 3, on the other hand did not inhibit WCE cleavage activity. ( C ) Testing AP site cleavage activity in wild-type and D148 polymorph. The figure shows that the activity was similar in both wild-type and the D148E polymorph. ( D ) Inhibitory activity of compound 4 against the D148E polymorphic variant of APE1 is shown here. D148E was more sensitive to inhibition by compound 4 compared with wild type.
    Figure Legend Snippet: ( A ) AP endonuclease activity assays using 18-mer radiolabelled oligonucleotide substrates (see Materials and methods) were performed using HeLa whole-cell extracts (WCE). Compound 4 showed 93% inhibition of AP cleavage activity using WCE. ( B ) Compound 3, on the other hand did not inhibit WCE cleavage activity. ( C ) Testing AP site cleavage activity in wild-type and D148 polymorph. The figure shows that the activity was similar in both wild-type and the D148E polymorph. ( D ) Inhibitory activity of compound 4 against the D148E polymorphic variant of APE1 is shown here. D148E was more sensitive to inhibition by compound 4 compared with wild type.

    Techniques Used: Activity Assay, Inhibition, Variant Assay

    Kinetics analysis. To evaluate potential mechanism of action of APE1 inhibitor, kinetic analysis was performed. Lineweaver–Burk plots and kinetic parameters determined from eight independent data points (note: error bars are in some cases too small to see) for compound 4 is shown here. The APE1 inhibitor was tested at three dose levels (5, 10 and 20 μ ) and oligonucleotide substrate was evaluated at three different concentrations (100, 200 and 500 n). The reaction was performed as described in methods. K M and k cat decreased at each inhibitor concentration (compared with no inhibitor) and the k cat /K M decreased at increasing inhibitor concentration. The data is consistent with uncompetitive inhibition.
    Figure Legend Snippet: Kinetics analysis. To evaluate potential mechanism of action of APE1 inhibitor, kinetic analysis was performed. Lineweaver–Burk plots and kinetic parameters determined from eight independent data points (note: error bars are in some cases too small to see) for compound 4 is shown here. The APE1 inhibitor was tested at three dose levels (5, 10 and 20 μ ) and oligonucleotide substrate was evaluated at three different concentrations (100, 200 and 500 n). The reaction was performed as described in methods. K M and k cat decreased at each inhibitor concentration (compared with no inhibitor) and the k cat /K M decreased at increasing inhibitor concentration. The data is consistent with uncompetitive inhibition.

    Techniques Used: Concentration Assay, Inhibition

    ( A ) Consensus score plot was constructed by plotting Gold Score ( x -axis) and Chem Score ( y -axis) for the 1679 virtual APE1 inhibitor candidates. The top ranking 25% of the compounds were shortlisted from the consensus plot and subjected to detailed biochemical analyses. ( B ) Primary screening. Fluorescence-based APE1 cleavage assay is shown here. If the DNA is cleaved at the abasic site at position 7 from the 5′ end by APE1, the 6-mer fluorescein-containing product will dissociate from its complement by thermal melting. As a result, the quenching effect of the 3′ dabcyl (which absorbs fluorescein fluorescence when in close proximity) is lost, and APE1 activity is measured indirectly as an increase in fluorescence signal. For detailed protocol see Materials and methods section. ( C ) APE1 inhibition by CRT0044876 is shown here. Control=no APE1 in reaction. ( D ) APE1 inhibition by compound 4 is shown here (IC 50 =11 μ ).
    Figure Legend Snippet: ( A ) Consensus score plot was constructed by plotting Gold Score ( x -axis) and Chem Score ( y -axis) for the 1679 virtual APE1 inhibitor candidates. The top ranking 25% of the compounds were shortlisted from the consensus plot and subjected to detailed biochemical analyses. ( B ) Primary screening. Fluorescence-based APE1 cleavage assay is shown here. If the DNA is cleaved at the abasic site at position 7 from the 5′ end by APE1, the 6-mer fluorescein-containing product will dissociate from its complement by thermal melting. As a result, the quenching effect of the 3′ dabcyl (which absorbs fluorescein fluorescence when in close proximity) is lost, and APE1 activity is measured indirectly as an increase in fluorescence signal. For detailed protocol see Materials and methods section. ( C ) APE1 inhibition by CRT0044876 is shown here. Control=no APE1 in reaction. ( D ) APE1 inhibition by compound 4 is shown here (IC 50 =11 μ ).

    Techniques Used: Construct, Fluorescence, Cleavage Assay, Activity Assay, Inhibition

    ( A ) APE1 expression in melanoma cell lines is shown here. ( B ) Inhibitor alone at 10 μ was relatively nontoxic to cells (as shown in Figure 7D ). We took the survival fraction as 100%. The percentage survival for those cells exposed to both inhibitor and temozolomide was plotted as a relative survival to cells exposed to the inhibitor alone. Potentiation of cytotoxicity of MMS by compound 4 (10 μ ) in SK-Mel30 cell line is shown here. ( C ) Potentiation of temozolomide by compound 4(10 μ ) in SK-Mel30 cell line is shown here. ( D ) Toxicity of compound 4 in HUVEC, SK-Mel30 and U89MG is shown here. Compound 4 was relatively nontoxic to HUVEC cells.
    Figure Legend Snippet: ( A ) APE1 expression in melanoma cell lines is shown here. ( B ) Inhibitor alone at 10 μ was relatively nontoxic to cells (as shown in Figure 7D ). We took the survival fraction as 100%. The percentage survival for those cells exposed to both inhibitor and temozolomide was plotted as a relative survival to cells exposed to the inhibitor alone. Potentiation of cytotoxicity of MMS by compound 4 (10 μ ) in SK-Mel30 cell line is shown here. ( C ) Potentiation of temozolomide by compound 4(10 μ ) in SK-Mel30 cell line is shown here. ( D ) Toxicity of compound 4 in HUVEC, SK-Mel30 and U89MG is shown here. Compound 4 was relatively nontoxic to HUVEC cells.

    Techniques Used: Expressing

    Secondary biochemical screening. ( A ) Fluorescence-based endonuclease IV cleavage assay is shown here. Compound 4 was tested at 100 μ and showed no inhibition of endonuclease IV. Control=no endonuclease IV in reaction ( B ). Compound 7 was tested at 100 μ and showed complete inhibition of endonuclease IV. Control=no endonuclease IV in reaction ( C ) Fluorescence queching assay did not reveal any quenching effect by compound 4. Control=no inhibitor in reaction ( D ). Radiolabelled assay showing inhibition of AP-site cleavage by APE1. Absence of 8-mer lower band indicates APE1 inhibition. See Material and methods for protcol details.
    Figure Legend Snippet: Secondary biochemical screening. ( A ) Fluorescence-based endonuclease IV cleavage assay is shown here. Compound 4 was tested at 100 μ and showed no inhibition of endonuclease IV. Control=no endonuclease IV in reaction ( B ). Compound 7 was tested at 100 μ and showed complete inhibition of endonuclease IV. Control=no endonuclease IV in reaction ( C ) Fluorescence queching assay did not reveal any quenching effect by compound 4. Control=no inhibitor in reaction ( D ). Radiolabelled assay showing inhibition of AP-site cleavage by APE1. Absence of 8-mer lower band indicates APE1 inhibition. See Material and methods for protcol details.

    Techniques Used: Fluorescence, Cleavage Assay, Inhibition

    Molecular Modelling. ( A ) The DNA repair domain active site was localised on the basis of the previously reported 10 critical amino acid residues that are essential for the AP endonuclease activity of APE1 (D70, D90, E96, Y171, D210, N212, D219, D283, D308 and H309, see text for details). Visual Molecular Dynamics1.8.7 was used to visualise the crystal structure of APE1. The molecular surface in the region of the V-shaped active site cleft is shown here. ( B ) Sybyl8.0 was used to build inhibitor templates. Chemical structures and docked poses of the four prototypical ligands onto APE1 active site are shown here.
    Figure Legend Snippet: Molecular Modelling. ( A ) The DNA repair domain active site was localised on the basis of the previously reported 10 critical amino acid residues that are essential for the AP endonuclease activity of APE1 (D70, D90, E96, Y171, D210, N212, D219, D283, D308 and H309, see text for details). Visual Molecular Dynamics1.8.7 was used to visualise the crystal structure of APE1. The molecular surface in the region of the V-shaped active site cleft is shown here. ( B ) Sybyl8.0 was used to build inhibitor templates. Chemical structures and docked poses of the four prototypical ligands onto APE1 active site are shown here.

    Techniques Used: Activity Assay

    ( A ) APE1 expression in glioma cell lines is shown here. ( B ) (1 and 2). Aldehyde reactive probe assay. U89MG cells were pretreated with 10 μ of compound 4 alone or MMS (600 μ ) or a combination of compound 4 and MMS. Genomic DNA extracted at 90 min for AP-site quatification. The combination treatment led to increased AP site content in the genomic DNA. ( C ) Inhibitor alone at 10 μ was relatively nontoxic to cells (as shown in Figure 7D ). We took the survival fraction as 100%. The percentage survival for those cells exposed to both inhibitor and temozolomide was plotted as a relative survival to cells exposed to the inhibitor alone. Potentiation of cytotoxicity of MMS by compound 4 (10 μ ) in U89 MG cell line is shown here. ( D ) Potentiation of temozolomide by compound 4 (10 μ ) in U89MG cell line is shown here.
    Figure Legend Snippet: ( A ) APE1 expression in glioma cell lines is shown here. ( B ) (1 and 2). Aldehyde reactive probe assay. U89MG cells were pretreated with 10 μ of compound 4 alone or MMS (600 μ ) or a combination of compound 4 and MMS. Genomic DNA extracted at 90 min for AP-site quatification. The combination treatment led to increased AP site content in the genomic DNA. ( C ) Inhibitor alone at 10 μ was relatively nontoxic to cells (as shown in Figure 7D ). We took the survival fraction as 100%. The percentage survival for those cells exposed to both inhibitor and temozolomide was plotted as a relative survival to cells exposed to the inhibitor alone. Potentiation of cytotoxicity of MMS by compound 4 (10 μ ) in U89 MG cell line is shown here. ( D ) Potentiation of temozolomide by compound 4 (10 μ ) in U89MG cell line is shown here.

    Techniques Used: Expressing

    15) Product Images from "Incidence and persistence of 8-oxo-7,8-dihydroguanine within a hairpin intermediate exacerbates a toxic oxidation cycle associated with trinucleotide repeat expansion"

    Article Title: Incidence and persistence of 8-oxo-7,8-dihydroguanine within a hairpin intermediate exacerbates a toxic oxidation cycle associated with trinucleotide repeat expansion

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2011.06.003

    Data obtained under multiple-turnover conditions for determination of k 3 . Shown is a graph of concentration of product as a function of time for (A) Mixed-DUP, (B) Loop-DUP, (C) Stem-DUP, and (D) Stem-HP. Conditions were 50 nM DNA, 5 nM hOGG1 or 5 nM hOGG1/50 nM APE1 in 20 mM Tris-HCl, 70 mM NaCl, 2 mM MgCl 2 , 100 µg/mL BSA, pH 7.6.
    Figure Legend Snippet: Data obtained under multiple-turnover conditions for determination of k 3 . Shown is a graph of concentration of product as a function of time for (A) Mixed-DUP, (B) Loop-DUP, (C) Stem-DUP, and (D) Stem-HP. Conditions were 50 nM DNA, 5 nM hOGG1 or 5 nM hOGG1/50 nM APE1 in 20 mM Tris-HCl, 70 mM NaCl, 2 mM MgCl 2 , 100 µg/mL BSA, pH 7.6.

    Techniques Used: Concentration Assay

    16) Product Images from "Incidence and persistence of 8-oxo-7,8-dihydroguanine within a hairpin intermediate exacerbates a toxic oxidation cycle associated with trinucleotide repeat expansion"

    Article Title: Incidence and persistence of 8-oxo-7,8-dihydroguanine within a hairpin intermediate exacerbates a toxic oxidation cycle associated with trinucleotide repeat expansion

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2011.06.003

    Data obtained under multiple-turnover conditions for determination of k 3 . Shown is a graph of concentration of product as a function of time for (A) Mixed-DUP, (B) Loop-DUP, (C) Stem-DUP, and (D) Stem-HP. Conditions were 50 nM DNA, 5 nM hOGG1 or 5 nM hOGG1/50 nM APE1 in 20 mM Tris-HCl, 70 mM NaCl, 2 mM MgCl 2 , 100 µg/mL BSA, pH 7.6.
    Figure Legend Snippet: Data obtained under multiple-turnover conditions for determination of k 3 . Shown is a graph of concentration of product as a function of time for (A) Mixed-DUP, (B) Loop-DUP, (C) Stem-DUP, and (D) Stem-HP. Conditions were 50 nM DNA, 5 nM hOGG1 or 5 nM hOGG1/50 nM APE1 in 20 mM Tris-HCl, 70 mM NaCl, 2 mM MgCl 2 , 100 µg/mL BSA, pH 7.6.

    Techniques Used: Concentration Assay

    17) Product Images from "4-Pregnen-21-ol-3,20-dione-21-(4-bromobenzenesufonate) (NSC 88915) and Related Novel Steroid Derivatives as Tyrosyl-DNA Phosphodiesterase (Tdp1) Inhibitors"

    Article Title: 4-Pregnen-21-ol-3,20-dione-21-(4-bromobenzenesufonate) (NSC 88915) and Related Novel Steroid Derivatives as Tyrosyl-DNA Phosphodiesterase (Tdp1) Inhibitors

    Journal: Journal of medicinal chemistry

    doi: 10.1021/jm901061s

    Counter screening of 1 against apurinic/apyrimidinic endonuclease enzyme (APE1) and topoisomerase I (Top1). A) Schematic representation of the APE1 gel-based biochemical assay. APE1 hydrolyzes the DNA phosphodiester backbone 5′ of the abasic site
    Figure Legend Snippet: Counter screening of 1 against apurinic/apyrimidinic endonuclease enzyme (APE1) and topoisomerase I (Top1). A) Schematic representation of the APE1 gel-based biochemical assay. APE1 hydrolyzes the DNA phosphodiester backbone 5′ of the abasic site

    Techniques Used:

    18) Product Images from "4-Pregnen-21-ol-3,20-dione-21-(4-bromobenzenesufonate) (NSC 88915) and Related Novel Steroid Derivatives as Tyrosyl-DNA Phosphodiesterase (Tdp1) Inhibitors"

    Article Title: 4-Pregnen-21-ol-3,20-dione-21-(4-bromobenzenesufonate) (NSC 88915) and Related Novel Steroid Derivatives as Tyrosyl-DNA Phosphodiesterase (Tdp1) Inhibitors

    Journal: Journal of medicinal chemistry

    doi: 10.1021/jm901061s

    Counter screening of 1 against apurinic/apyrimidinic endonuclease enzyme (APE1) and topoisomerase I (Top1). A) Schematic representation of the APE1 gel-based biochemical assay. APE1 hydrolyzes the DNA phosphodiester backbone 5′ of the abasic site
    Figure Legend Snippet: Counter screening of 1 against apurinic/apyrimidinic endonuclease enzyme (APE1) and topoisomerase I (Top1). A) Schematic representation of the APE1 gel-based biochemical assay. APE1 hydrolyzes the DNA phosphodiester backbone 5′ of the abasic site

    Techniques Used:

    19) Product Images from "ETS transcription factors induce a unique UV damage signature that drives recurrent mutagenesis in melanoma"

    Article Title: ETS transcription factors induce a unique UV damage signature that drives recurrent mutagenesis in melanoma

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05064-0

    Genome-wide map of CPD lesions reveals that CPDs are elevated at active TFBS. a Schematic diagram of the CPD-seq method for mapping CPD lesions at single nucleotide resolution. ‘T = C′ indicates a CPD lesion at TC dipyrimidine. Oligonucleotide adapters are indicated in green and purple; ‘NNNNNN′ indicates a random DNA hexamer. A 3′ hydroxyl is indicated with OH, while ‘dd’ indicates a dideoxy 3′ end. The CPD lesion is cleaved with T4 endonuclease V and apurinic/apyrimidinic endonuclease (APE1) to generate a free 3′ hydroxyl immediately upstream of the CPD lesion, which is ligated to an adapter and sequenced. b Mutation density surrounding active promoter-proximal TFBS from 184 sequenced melanoma tumors 18 . Observed mutation density (i.e., in melanoma tumors) was analyzed adjacent to known TFBS located in promoter-proximal regions (up to 2500 bp upstream of transcription start site) that were considered active (i.e., overlapping with melanocyte DNase I-hypersensitivity (DHS) regions) for 82 distinct TFs. Expected mutation density was determined from the corresponding DNA sequences surrounding each active promoter-proximal TFBS, based on the trinucleotide mutation signature frequencies for all promoter-proximal regions. c Same as part ( b ), except mutations were analyzed adjacent to promoter-proximal TFBS that were considered inactive (i.e., not overlapping with melanocyte DHS regions). d Average number of CPD lesions (per TFBS) adjacent to active promoter-proximal TFBS. CPD lesions were mapped using CPD-seq from UV-irradiated NHF1 cells (100 J m −2 ) or isolated NHF1 DNA that was UV-irradiated (80 J m −2 ) in vitro (naked DNA). Cellular DNA was harvested immediately after UV irradiation, so essentially no repair was allowed to occur. e Same as in part ( d ), except CPD lesions were analyzed adjacent to inactive promoter-proximal TFBS
    Figure Legend Snippet: Genome-wide map of CPD lesions reveals that CPDs are elevated at active TFBS. a Schematic diagram of the CPD-seq method for mapping CPD lesions at single nucleotide resolution. ‘T = C′ indicates a CPD lesion at TC dipyrimidine. Oligonucleotide adapters are indicated in green and purple; ‘NNNNNN′ indicates a random DNA hexamer. A 3′ hydroxyl is indicated with OH, while ‘dd’ indicates a dideoxy 3′ end. The CPD lesion is cleaved with T4 endonuclease V and apurinic/apyrimidinic endonuclease (APE1) to generate a free 3′ hydroxyl immediately upstream of the CPD lesion, which is ligated to an adapter and sequenced. b Mutation density surrounding active promoter-proximal TFBS from 184 sequenced melanoma tumors 18 . Observed mutation density (i.e., in melanoma tumors) was analyzed adjacent to known TFBS located in promoter-proximal regions (up to 2500 bp upstream of transcription start site) that were considered active (i.e., overlapping with melanocyte DNase I-hypersensitivity (DHS) regions) for 82 distinct TFs. Expected mutation density was determined from the corresponding DNA sequences surrounding each active promoter-proximal TFBS, based on the trinucleotide mutation signature frequencies for all promoter-proximal regions. c Same as part ( b ), except mutations were analyzed adjacent to promoter-proximal TFBS that were considered inactive (i.e., not overlapping with melanocyte DHS regions). d Average number of CPD lesions (per TFBS) adjacent to active promoter-proximal TFBS. CPD lesions were mapped using CPD-seq from UV-irradiated NHF1 cells (100 J m −2 ) or isolated NHF1 DNA that was UV-irradiated (80 J m −2 ) in vitro (naked DNA). Cellular DNA was harvested immediately after UV irradiation, so essentially no repair was allowed to occur. e Same as in part ( d ), except CPD lesions were analyzed adjacent to inactive promoter-proximal TFBS

    Techniques Used: Genome Wide, Mutagenesis, Irradiation, Isolation, In Vitro

    20) Product Images from "The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes *"

    Article Title: The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.441444

    Assessment of the removal of rotationally and translationally positioned uracils by UDG and APE1. A, NCPs containing a single uracil at different sites were incubated with UDG and APE1. Open symbols represent in uracils as follows: red square , NCP-UI
    Figure Legend Snippet: Assessment of the removal of rotationally and translationally positioned uracils by UDG and APE1. A, NCPs containing a single uracil at different sites were incubated with UDG and APE1. Open symbols represent in uracils as follows: red square , NCP-UI

    Techniques Used: Incubation

    UDG and APE1 Digestion
    Figure Legend Snippet: UDG and APE1 Digestion

    Techniques Used:

    Polymerase β extension activity in NCPs near the dyad. A, representative gels for NCP-gO (+10) and NCP-gI (+4) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (+10) and NCP-gI (+4) were incubated with pol β and APE1
    Figure Legend Snippet: Polymerase β extension activity in NCPs near the dyad. A, representative gels for NCP-gO (+10) and NCP-gI (+4) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (+10) and NCP-gI (+4) were incubated with pol β and APE1

    Techniques Used: Activity Assay, Incubation

    Polymerase β extension activity in NCPs near DNA ends. A, representative gels for NCP-gO (−35) and NCP-gI (−49) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (−35) and NCP-gI (−49) were incubated
    Figure Legend Snippet: Polymerase β extension activity in NCPs near DNA ends. A, representative gels for NCP-gO (−35) and NCP-gI (−49) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (−35) and NCP-gI (−49) were incubated

    Techniques Used: Activity Assay, Incubation

    UDG and APE1 Digestion
    Figure Legend Snippet: UDG and APE1 Digestion

    Techniques Used:

    21) Product Images from "The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes *"

    Article Title: The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.441444

    Assessment of the removal of rotationally and translationally positioned uracils by UDG and APE1. A, NCPs containing a single uracil at different sites were incubated with UDG and APE1. Open symbols represent in uracils as follows: red square , NCP-UI
    Figure Legend Snippet: Assessment of the removal of rotationally and translationally positioned uracils by UDG and APE1. A, NCPs containing a single uracil at different sites were incubated with UDG and APE1. Open symbols represent in uracils as follows: red square , NCP-UI

    Techniques Used: Incubation

    UDG and APE1 Digestion
    Figure Legend Snippet: UDG and APE1 Digestion

    Techniques Used:

    Polymerase β extension activity in NCPs near the dyad. A, representative gels for NCP-gO (+10) and NCP-gI (+4) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (+10) and NCP-gI (+4) were incubated with pol β and APE1
    Figure Legend Snippet: Polymerase β extension activity in NCPs near the dyad. A, representative gels for NCP-gO (+10) and NCP-gI (+4) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (+10) and NCP-gI (+4) were incubated with pol β and APE1

    Techniques Used: Activity Assay, Incubation

    Polymerase β extension activity in NCPs near DNA ends. A, representative gels for NCP-gO (−35) and NCP-gI (−49) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (−35) and NCP-gI (−49) were incubated
    Figure Legend Snippet: Polymerase β extension activity in NCPs near DNA ends. A, representative gels for NCP-gO (−35) and NCP-gI (−49) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (−35) and NCP-gI (−49) were incubated

    Techniques Used: Activity Assay, Incubation

    UDG and APE1 Digestion
    Figure Legend Snippet: UDG and APE1 Digestion

    Techniques Used:

    22) Product Images from "Controllable Autocatalytic Cleavage-Mediated Fluorescence Recovery for Homogeneous Sensing of Alkyladenine DNA Glycosylase from Human Cancer Cells"

    Article Title: Controllable Autocatalytic Cleavage-Mediated Fluorescence Recovery for Homogeneous Sensing of Alkyladenine DNA Glycosylase from Human Cancer Cells

    Journal: Theranostics

    doi: 10.7150/thno.35393

    Dependence of initial velocity ( V ) on different concentrations of hairpin substrate. The 0.1 U/µL hAAG and 0.3 U/µL APE1 were used in this experiment. The time for hAAG-induced 2′-deoxyinosine excision reaction was 5 min. Error bars show the standard deviation of three experiments.
    Figure Legend Snippet: Dependence of initial velocity ( V ) on different concentrations of hairpin substrate. The 0.1 U/µL hAAG and 0.3 U/µL APE1 were used in this experiment. The time for hAAG-induced 2′-deoxyinosine excision reaction was 5 min. Error bars show the standard deviation of three experiments.

    Techniques Used: Standard Deviation

    (A) Nondenaturing PAGE analysis of the products of hAAG-catalyzed cleavage reaction under different reaction conditions. Lane M, the DNA ladder marker; lane 1, the synthesized cleavage product; lane 2, the synthesized trigger 1; lane 3, the synthesized HP1; lane 4, in the presence of APE1 + HP1; lane 5, in the presence of hAAG + APE1 + HP1. (B) Nondenaturing PAGE analysis of the products of T7 exo-assisted autocatalytic recycling cleavage reaction. Lane M, the DNA ladder marker; lane 1, in the absence of hAAG; lane 2, the synthesized trigger 2; lane 3, in the presence of hAAG. SYBR Gold is used as the fluorescent indicator. The 0.1 U/µL hAAG and 0.3 U/µL APE1 were used in this experiment.
    Figure Legend Snippet: (A) Nondenaturing PAGE analysis of the products of hAAG-catalyzed cleavage reaction under different reaction conditions. Lane M, the DNA ladder marker; lane 1, the synthesized cleavage product; lane 2, the synthesized trigger 1; lane 3, the synthesized HP1; lane 4, in the presence of APE1 + HP1; lane 5, in the presence of hAAG + APE1 + HP1. (B) Nondenaturing PAGE analysis of the products of T7 exo-assisted autocatalytic recycling cleavage reaction. Lane M, the DNA ladder marker; lane 1, in the absence of hAAG; lane 2, the synthesized trigger 2; lane 3, in the presence of hAAG. SYBR Gold is used as the fluorescent indicator. The 0.1 U/µL hAAG and 0.3 U/µL APE1 were used in this experiment.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Marker, Synthesized

    Schematic illustration of the controllable autocatalytic cleavage-induced fluorescence recovery for hAAG assay. This strategy involves two consecutive steps: (1) the specific cleavage of HP1 at 2′-deoxyinosine site by hAAG and APE1, and (2) T7 exo-assisted autocatalytic recycling signal amplification.
    Figure Legend Snippet: Schematic illustration of the controllable autocatalytic cleavage-induced fluorescence recovery for hAAG assay. This strategy involves two consecutive steps: (1) the specific cleavage of HP1 at 2′-deoxyinosine site by hAAG and APE1, and (2) T7 exo-assisted autocatalytic recycling signal amplification.

    Techniques Used: Fluorescence, Amplification

    Dependence of the relative activity of hAAG on different concentrations of CdCl 2 . The 0.1 U/µL hAAG and 0.3 U/µL APE1 were used in this experiment. The error bars represent the standard deviations of the three experiments.
    Figure Legend Snippet: Dependence of the relative activity of hAAG on different concentrations of CdCl 2 . The 0.1 U/µL hAAG and 0.3 U/µL APE1 were used in this experiment. The error bars represent the standard deviations of the three experiments.

    Techniques Used: Activity Assay

    Mechanism of hAAG-catalyzed base-excision repair. The hAAG can remove the damaged 2′-deoxyinosine (red color) to produce an AP site. The AP site will subsequently be cleaved by APE1 to generate the 5′-dRP and 3′-OH termini.
    Figure Legend Snippet: Mechanism of hAAG-catalyzed base-excision repair. The hAAG can remove the damaged 2′-deoxyinosine (red color) to produce an AP site. The AP site will subsequently be cleaved by APE1 to generate the 5′-dRP and 3′-OH termini.

    Techniques Used:

    23) Product Images from "Incidence and persistence of 8-oxo-7,8-dihydroguanine within a hairpin intermediate exacerbates a toxic oxidation cycle associated with trinucleotide repeat expansion"

    Article Title: Incidence and persistence of 8-oxo-7,8-dihydroguanine within a hairpin intermediate exacerbates a toxic oxidation cycle associated with trinucleotide repeat expansion

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2011.06.003

    Data obtained under multiple-turnover conditions for determination of k 3 . Shown is a graph of concentration of product as a function of time for (A) Mixed-DUP, (B) Loop-DUP, (C) Stem-DUP, and (D) Stem-HP. Conditions were 50 nM DNA, 5 nM hOGG1 or 5 nM hOGG1/50 nM APE1 in 20 mM Tris-HCl, 70 mM NaCl, 2 mM MgCl 2 , 100 µg/mL BSA, pH 7.6.
    Figure Legend Snippet: Data obtained under multiple-turnover conditions for determination of k 3 . Shown is a graph of concentration of product as a function of time for (A) Mixed-DUP, (B) Loop-DUP, (C) Stem-DUP, and (D) Stem-HP. Conditions were 50 nM DNA, 5 nM hOGG1 or 5 nM hOGG1/50 nM APE1 in 20 mM Tris-HCl, 70 mM NaCl, 2 mM MgCl 2 , 100 µg/mL BSA, pH 7.6.

    Techniques Used: Concentration Assay

    24) Product Images from "The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes *"

    Article Title: The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.441444

    Assessment of the removal of rotationally and translationally positioned uracils by UDG and APE1. A, NCPs containing a single uracil at different sites were incubated with UDG and APE1. Open symbols represent in uracils as follows: red square , NCP-UI
    Figure Legend Snippet: Assessment of the removal of rotationally and translationally positioned uracils by UDG and APE1. A, NCPs containing a single uracil at different sites were incubated with UDG and APE1. Open symbols represent in uracils as follows: red square , NCP-UI

    Techniques Used: Incubation

    UDG and APE1 Digestion
    Figure Legend Snippet: UDG and APE1 Digestion

    Techniques Used:

    Polymerase β extension activity in NCPs near the dyad. A, representative gels for NCP-gO (+10) and NCP-gI (+4) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (+10) and NCP-gI (+4) were incubated with pol β and APE1
    Figure Legend Snippet: Polymerase β extension activity in NCPs near the dyad. A, representative gels for NCP-gO (+10) and NCP-gI (+4) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (+10) and NCP-gI (+4) were incubated with pol β and APE1

    Techniques Used: Activity Assay, Incubation

    Polymerase β extension activity in NCPs near DNA ends. A, representative gels for NCP-gO (−35) and NCP-gI (−49) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (−35) and NCP-gI (−49) were incubated
    Figure Legend Snippet: Polymerase β extension activity in NCPs near DNA ends. A, representative gels for NCP-gO (−35) and NCP-gI (−49) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (−35) and NCP-gI (−49) were incubated

    Techniques Used: Activity Assay, Incubation

    UDG and APE1 Digestion
    Figure Legend Snippet: UDG and APE1 Digestion

    Techniques Used:

    25) Product Images from "The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes *"

    Article Title: The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.441444

    Assessment of the removal of rotationally and translationally positioned uracils by UDG and APE1. A, NCPs containing a single uracil at different sites were incubated with UDG and APE1. Open symbols represent in uracils as follows: red square , NCP-UI
    Figure Legend Snippet: Assessment of the removal of rotationally and translationally positioned uracils by UDG and APE1. A, NCPs containing a single uracil at different sites were incubated with UDG and APE1. Open symbols represent in uracils as follows: red square , NCP-UI

    Techniques Used: Incubation

    UDG and APE1 Digestion
    Figure Legend Snippet: UDG and APE1 Digestion

    Techniques Used:

    Polymerase β extension activity in NCPs near the dyad. A, representative gels for NCP-gO (+10) and NCP-gI (+4) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (+10) and NCP-gI (+4) were incubated with pol β and APE1
    Figure Legend Snippet: Polymerase β extension activity in NCPs near the dyad. A, representative gels for NCP-gO (+10) and NCP-gI (+4) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (+10) and NCP-gI (+4) were incubated with pol β and APE1

    Techniques Used: Activity Assay, Incubation

    Polymerase β extension activity in NCPs near DNA ends. A, representative gels for NCP-gO (−35) and NCP-gI (−49) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (−35) and NCP-gI (−49) were incubated
    Figure Legend Snippet: Polymerase β extension activity in NCPs near DNA ends. A, representative gels for NCP-gO (−35) and NCP-gI (−49) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (−35) and NCP-gI (−49) were incubated

    Techniques Used: Activity Assay, Incubation

    UDG and APE1 Digestion
    Figure Legend Snippet: UDG and APE1 Digestion

    Techniques Used:

    26) Product Images from "Controllable Autocatalytic Cleavage-Mediated Fluorescence Recovery for Homogeneous Sensing of Alkyladenine DNA Glycosylase from Human Cancer Cells"

    Article Title: Controllable Autocatalytic Cleavage-Mediated Fluorescence Recovery for Homogeneous Sensing of Alkyladenine DNA Glycosylase from Human Cancer Cells

    Journal: Theranostics

    doi: 10.7150/thno.35393

    Dependence of initial velocity ( V ) on different concentrations of hairpin substrate. The 0.1 U/µL hAAG and 0.3 U/µL APE1 were used in this experiment. The time for hAAG-induced 2′-deoxyinosine excision reaction was 5 min. Error bars show the standard deviation of three experiments.
    Figure Legend Snippet: Dependence of initial velocity ( V ) on different concentrations of hairpin substrate. The 0.1 U/µL hAAG and 0.3 U/µL APE1 were used in this experiment. The time for hAAG-induced 2′-deoxyinosine excision reaction was 5 min. Error bars show the standard deviation of three experiments.

    Techniques Used: Standard Deviation

    (A) Nondenaturing PAGE analysis of the products of hAAG-catalyzed cleavage reaction under different reaction conditions. Lane M, the DNA ladder marker; lane 1, the synthesized cleavage product; lane 2, the synthesized trigger 1; lane 3, the synthesized HP1; lane 4, in the presence of APE1 + HP1; lane 5, in the presence of hAAG + APE1 + HP1. (B) Nondenaturing PAGE analysis of the products of T7 exo-assisted autocatalytic recycling cleavage reaction. Lane M, the DNA ladder marker; lane 1, in the absence of hAAG; lane 2, the synthesized trigger 2; lane 3, in the presence of hAAG. SYBR Gold is used as the fluorescent indicator. The 0.1 U/µL hAAG and 0.3 U/µL APE1 were used in this experiment.
    Figure Legend Snippet: (A) Nondenaturing PAGE analysis of the products of hAAG-catalyzed cleavage reaction under different reaction conditions. Lane M, the DNA ladder marker; lane 1, the synthesized cleavage product; lane 2, the synthesized trigger 1; lane 3, the synthesized HP1; lane 4, in the presence of APE1 + HP1; lane 5, in the presence of hAAG + APE1 + HP1. (B) Nondenaturing PAGE analysis of the products of T7 exo-assisted autocatalytic recycling cleavage reaction. Lane M, the DNA ladder marker; lane 1, in the absence of hAAG; lane 2, the synthesized trigger 2; lane 3, in the presence of hAAG. SYBR Gold is used as the fluorescent indicator. The 0.1 U/µL hAAG and 0.3 U/µL APE1 were used in this experiment.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Marker, Synthesized

    Schematic illustration of the controllable autocatalytic cleavage-induced fluorescence recovery for hAAG assay. This strategy involves two consecutive steps: (1) the specific cleavage of HP1 at 2′-deoxyinosine site by hAAG and APE1, and (2) T7 exo-assisted autocatalytic recycling signal amplification.
    Figure Legend Snippet: Schematic illustration of the controllable autocatalytic cleavage-induced fluorescence recovery for hAAG assay. This strategy involves two consecutive steps: (1) the specific cleavage of HP1 at 2′-deoxyinosine site by hAAG and APE1, and (2) T7 exo-assisted autocatalytic recycling signal amplification.

    Techniques Used: Fluorescence, Amplification

    Dependence of the relative activity of hAAG on different concentrations of CdCl 2 . The 0.1 U/µL hAAG and 0.3 U/µL APE1 were used in this experiment. The error bars represent the standard deviations of the three experiments.
    Figure Legend Snippet: Dependence of the relative activity of hAAG on different concentrations of CdCl 2 . The 0.1 U/µL hAAG and 0.3 U/µL APE1 were used in this experiment. The error bars represent the standard deviations of the three experiments.

    Techniques Used: Activity Assay

    Mechanism of hAAG-catalyzed base-excision repair. The hAAG can remove the damaged 2′-deoxyinosine (red color) to produce an AP site. The AP site will subsequently be cleaved by APE1 to generate the 5′-dRP and 3′-OH termini.
    Figure Legend Snippet: Mechanism of hAAG-catalyzed base-excision repair. The hAAG can remove the damaged 2′-deoxyinosine (red color) to produce an AP site. The AP site will subsequently be cleaved by APE1 to generate the 5′-dRP and 3′-OH termini.

    Techniques Used:

    27) Product Images from "Alzheimer’s Disease Associated Polymorphisms in Human OGG1 Alter Catalytic Activity and Sensitize Cells to DNA Damage"

    Article Title: Alzheimer’s Disease Associated Polymorphisms in Human OGG1 Alter Catalytic Activity and Sensitize Cells to DNA Damage

    Journal: Free radical biology & medicine

    doi: 10.1016/j.freeradbiomed.2013.05.010

    Decreased AP lyase activity and differential stimulation of polymorphic OGG1s by APE1 (A) OGG1 recombinant proteins (64nM) were incubated at 37°C for 15 minutes in the presence (+) or absence (−) of 45pM APE1, along with a 5′-end-labeled oligonucleotide duplex containing an 8-oxoG lesion. The cleavage products were analyzed on a 15% polyacrylamide gel containing 7M Urea and imaged on a phosphorimager. A representative experiment is shown. (B) The histogram represents the mean ± S.E.M. from five independent experiments. The incision value was calculated by taking the amount of cleaved substrate (lower band) normalized to the amount of cleaved + uncleaved substrate (lower + upper bands) . *p
    Figure Legend Snippet: Decreased AP lyase activity and differential stimulation of polymorphic OGG1s by APE1 (A) OGG1 recombinant proteins (64nM) were incubated at 37°C for 15 minutes in the presence (+) or absence (−) of 45pM APE1, along with a 5′-end-labeled oligonucleotide duplex containing an 8-oxoG lesion. The cleavage products were analyzed on a 15% polyacrylamide gel containing 7M Urea and imaged on a phosphorimager. A representative experiment is shown. (B) The histogram represents the mean ± S.E.M. from five independent experiments. The incision value was calculated by taking the amount of cleaved substrate (lower band) normalized to the amount of cleaved + uncleaved substrate (lower + upper bands) . *p

    Techniques Used: Activity Assay, Recombinant, Incubation, Labeling

    28) Product Images from "Alzheimer’s Disease Associated Polymorphisms in Human OGG1 Alter Catalytic Activity and Sensitize Cells to DNA Damage"

    Article Title: Alzheimer’s Disease Associated Polymorphisms in Human OGG1 Alter Catalytic Activity and Sensitize Cells to DNA Damage

    Journal: Free radical biology & medicine

    doi: 10.1016/j.freeradbiomed.2013.05.010

    Decreased AP lyase activity and differential stimulation of polymorphic OGG1s by APE1 (A) OGG1 recombinant proteins (64nM) were incubated at 37°C for 15 minutes in the presence (+) or absence (−) of 45pM APE1, along with a 5′-end-labeled oligonucleotide duplex containing an 8-oxoG lesion. The cleavage products were analyzed on a 15% polyacrylamide gel containing 7M Urea and imaged on a phosphorimager. A representative experiment is shown. (B) The histogram represents the mean ± S.E.M. from five independent experiments. The incision value was calculated by taking the amount of cleaved substrate (lower band) normalized to the amount of cleaved + uncleaved substrate (lower + upper bands) . *p
    Figure Legend Snippet: Decreased AP lyase activity and differential stimulation of polymorphic OGG1s by APE1 (A) OGG1 recombinant proteins (64nM) were incubated at 37°C for 15 minutes in the presence (+) or absence (−) of 45pM APE1, along with a 5′-end-labeled oligonucleotide duplex containing an 8-oxoG lesion. The cleavage products were analyzed on a 15% polyacrylamide gel containing 7M Urea and imaged on a phosphorimager. A representative experiment is shown. (B) The histogram represents the mean ± S.E.M. from five independent experiments. The incision value was calculated by taking the amount of cleaved substrate (lower band) normalized to the amount of cleaved + uncleaved substrate (lower + upper bands) . *p

    Techniques Used: Activity Assay, Recombinant, Incubation, Labeling

    29) Product Images from "Alzheimer’s Disease Associated Polymorphisms in Human OGG1 Alter Catalytic Activity and Sensitize Cells to DNA Damage"

    Article Title: Alzheimer’s Disease Associated Polymorphisms in Human OGG1 Alter Catalytic Activity and Sensitize Cells to DNA Damage

    Journal: Free radical biology & medicine

    doi: 10.1016/j.freeradbiomed.2013.05.010

    Decreased AP lyase activity and differential stimulation of polymorphic OGG1s by APE1 (A) OGG1 recombinant proteins (64nM) were incubated at 37°C for 15 minutes in the presence (+) or absence (−) of 45pM APE1, along with a 5′-end-labeled oligonucleotide duplex containing an 8-oxoG lesion. The cleavage products were analyzed on a 15% polyacrylamide gel containing 7M Urea and imaged on a phosphorimager. A representative experiment is shown. (B) The histogram represents the mean ± S.E.M. from five independent experiments. The incision value was calculated by taking the amount of cleaved substrate (lower band) normalized to the amount of cleaved + uncleaved substrate (lower + upper bands) . *p
    Figure Legend Snippet: Decreased AP lyase activity and differential stimulation of polymorphic OGG1s by APE1 (A) OGG1 recombinant proteins (64nM) were incubated at 37°C for 15 minutes in the presence (+) or absence (−) of 45pM APE1, along with a 5′-end-labeled oligonucleotide duplex containing an 8-oxoG lesion. The cleavage products were analyzed on a 15% polyacrylamide gel containing 7M Urea and imaged on a phosphorimager. A representative experiment is shown. (B) The histogram represents the mean ± S.E.M. from five independent experiments. The incision value was calculated by taking the amount of cleaved substrate (lower band) normalized to the amount of cleaved + uncleaved substrate (lower + upper bands) . *p

    Techniques Used: Activity Assay, Recombinant, Incubation, Labeling

    30) Product Images from "Enhanced activity of adenine-DNA glycosylase (Myh) by apurinic/apyrimidinic endonuclease (Ape1) in mammalian base excision repair of an A/GO mismatch"

    Article Title: Enhanced activity of adenine-DNA glycosylase (Myh) by apurinic/apyrimidinic endonuclease (Ape1) in mammalian base excision repair of an A/GO mismatch

    Journal: Nucleic Acids Research

    doi:

    Turnover of Myh. The glycosylase reactions were carried out at 37°C with 5 nM A/GO substrate and 0.5 nM Myh in the presence of 5 nM Ape1 or buffer. Samples were taken at different time points, with or without alkaline treatment, and analyzed on a 15% denaturing polyacrylamide gel. A plot of the formation of AP/GO with alkaline treatment (+NaOH) or cAP/GO without alkaline treatment (–NaOH) versus time is shown.
    Figure Legend Snippet: Turnover of Myh. The glycosylase reactions were carried out at 37°C with 5 nM A/GO substrate and 0.5 nM Myh in the presence of 5 nM Ape1 or buffer. Samples were taken at different time points, with or without alkaline treatment, and analyzed on a 15% denaturing polyacrylamide gel. A plot of the formation of AP/GO with alkaline treatment (+NaOH) or cAP/GO without alkaline treatment (–NaOH) versus time is shown.

    Techniques Used:

    Substrate preference of Myh and Myh/Ape1. Substrate DNA (0.2 nM) was incubated with Myh alone or Myh/Ape1 at a constant molar ratio of 1:100. The glycosylase reaction was carried out at 37°C for 30 min. Then each reaction mixture, with alkaline treatment, was analyzed on a 15% denaturing polyacrylamide gel. An asterisk indicates the 5′-end-labeled strand. ( A ) Plot of the fraction of glycosylase product versus Myh. Each individual point is represented with error bars as an average of data from two to four experiments. ( B ) Plot of the fraction of glycosylase product versus Myh/Ape1 (only Myh concentrations are indicated; Ape1 concentrations can be calculated based on a constant 1:100 molar ratio).
    Figure Legend Snippet: Substrate preference of Myh and Myh/Ape1. Substrate DNA (0.2 nM) was incubated with Myh alone or Myh/Ape1 at a constant molar ratio of 1:100. The glycosylase reaction was carried out at 37°C for 30 min. Then each reaction mixture, with alkaline treatment, was analyzed on a 15% denaturing polyacrylamide gel. An asterisk indicates the 5′-end-labeled strand. ( A ) Plot of the fraction of glycosylase product versus Myh. Each individual point is represented with error bars as an average of data from two to four experiments. ( B ) Plot of the fraction of glycosylase product versus Myh/Ape1 (only Myh concentrations are indicated; Ape1 concentrations can be calculated based on a constant 1:100 molar ratio).

    Techniques Used: Incubation, Labeling

    Accumulation of the glycosylase product AP/GO (A) and the cleaved product cAP/GO (B) over time. The glycosylase reactions were carried out at 37°C with 0.2 nM A/GO substrate and 0.5 nM Myh in the presence of 5 nM wild-type Ape1 (wt Ape1), 5 nM catalytic mutant (H309N mutant) or buffer. Samples were taken at different time points as indicated, with or without alkaline treatment, and were analyzed on a 15% denaturing polyacrylamide gel. ( A ) Plot of the fraction of glycosylase product AP/GO versus time. Individual points represented with error bars are the average from two experiments. ( B ) Plot of the fraction of cleaved product cAP/GO versus time.
    Figure Legend Snippet: Accumulation of the glycosylase product AP/GO (A) and the cleaved product cAP/GO (B) over time. The glycosylase reactions were carried out at 37°C with 0.2 nM A/GO substrate and 0.5 nM Myh in the presence of 5 nM wild-type Ape1 (wt Ape1), 5 nM catalytic mutant (H309N mutant) or buffer. Samples were taken at different time points as indicated, with or without alkaline treatment, and were analyzed on a 15% denaturing polyacrylamide gel. ( A ) Plot of the fraction of glycosylase product AP/GO versus time. Individual points represented with error bars are the average from two experiments. ( B ) Plot of the fraction of cleaved product cAP/GO versus time.

    Techniques Used: Mutagenesis

    Stimulation of Myh binding to non-cleavable T/GO substrate by Ape1 and H309N. The binding reactions were carried out at 37°C for 10 min with 50 pM T/GO DNA in the presence of Myh, Myh/Ape1, Myh/H309N or Myh/BSA at the concentrations indicated. The binding products were analyzed by EMSA on an 8% native polyacrylamide gel. An asterisk indicates the 5′-end-labeled strand. The observed bands a and b are marked on the right. The bound and free DNA forms are indicated on the left.
    Figure Legend Snippet: Stimulation of Myh binding to non-cleavable T/GO substrate by Ape1 and H309N. The binding reactions were carried out at 37°C for 10 min with 50 pM T/GO DNA in the presence of Myh, Myh/Ape1, Myh/H309N or Myh/BSA at the concentrations indicated. The binding products were analyzed by EMSA on an 8% native polyacrylamide gel. An asterisk indicates the 5′-end-labeled strand. The observed bands a and b are marked on the right. The bound and free DNA forms are indicated on the left.

    Techniques Used: Binding Assay, Labeling

    Cleavage of AP DNA by Ape1 and its catalytic mutant H309N. The glycosylase reactions were carried out at 37°C for 30 min with 0.2 nM A/GO substrate, 5 nM Myh and increasing amounts of Ape1 as indicated. Then each reaction mixture, without alkaline treatment, was analyzed on a 15% denaturing polyacrylamide gel. A plot of the fraction cleaved by wild-type Ape1 or catalytic mutant H309N versus concentration of Ape1 protein is shown.
    Figure Legend Snippet: Cleavage of AP DNA by Ape1 and its catalytic mutant H309N. The glycosylase reactions were carried out at 37°C for 30 min with 0.2 nM A/GO substrate, 5 nM Myh and increasing amounts of Ape1 as indicated. Then each reaction mixture, without alkaline treatment, was analyzed on a 15% denaturing polyacrylamide gel. A plot of the fraction cleaved by wild-type Ape1 or catalytic mutant H309N versus concentration of Ape1 protein is shown.

    Techniques Used: Mutagenesis, Concentration Assay

    Stimulation of Myh–DNA complex formation by Ape1 and H309N. The glycosylase reactions were carried out at 37°C for 10 min with 0.2 nM A/GO substrate and 0.5 nM Myh in the presence of 50 nM wild-type Ape1 (wt Ape1), 50 nM catalytic mutant H309N (H309N mutant), 50 nM BSA or buffer. One aliquot of each reaction was directly analyzed by EMSA on an 8% native polyacrylamide gel. The other aliquot, with alkaline treatment, was analyzed on a 15% denaturing polyacrylamide gel. An asterisk indicates the 5′-end-labeled strand. ( A ) Phosphoimage of Myh–DNA complexes. The observed bands a and b are marked on the right. The bound and free DNA forms are indicated on the left. ( B ) Phosphoimage of accumulation of glycosylase product. The intact 96mer DNA (Intact) and cleaved 60mer product (Cleaved product) are indicated on the left.
    Figure Legend Snippet: Stimulation of Myh–DNA complex formation by Ape1 and H309N. The glycosylase reactions were carried out at 37°C for 10 min with 0.2 nM A/GO substrate and 0.5 nM Myh in the presence of 50 nM wild-type Ape1 (wt Ape1), 50 nM catalytic mutant H309N (H309N mutant), 50 nM BSA or buffer. One aliquot of each reaction was directly analyzed by EMSA on an 8% native polyacrylamide gel. The other aliquot, with alkaline treatment, was analyzed on a 15% denaturing polyacrylamide gel. An asterisk indicates the 5′-end-labeled strand. ( A ) Phosphoimage of Myh–DNA complexes. The observed bands a and b are marked on the right. The bound and free DNA forms are indicated on the left. ( B ) Phosphoimage of accumulation of glycosylase product. The intact 96mer DNA (Intact) and cleaved 60mer product (Cleaved product) are indicated on the left.

    Techniques Used: Mutagenesis, Labeling

    Accumulation of free cAP/GO and subsequent binding to Myh. The glycosylase reactions were carried out at 37°C for 30 min with 0.2 nM A/GO substrate and ( A ) 0.5 nM Myh (sub-saturating concentration) in the presence of increasing amounts of Ape1 or ( B ) 0.5 nM Myh/50 nM Ape1 with addition of more Myh after the 30 min glycosylase reaction. Myh–DNA complexes were analyzed by EMSA on an 8% native polyacrylamide gel. The observed bands a, b and bb are marked on the right. The bound and free DNA forms are indicated on the left. An asterisk indicates the 5′-end-labeled strand.
    Figure Legend Snippet: Accumulation of free cAP/GO and subsequent binding to Myh. The glycosylase reactions were carried out at 37°C for 30 min with 0.2 nM A/GO substrate and ( A ) 0.5 nM Myh (sub-saturating concentration) in the presence of increasing amounts of Ape1 or ( B ) 0.5 nM Myh/50 nM Ape1 with addition of more Myh after the 30 min glycosylase reaction. Myh–DNA complexes were analyzed by EMSA on an 8% native polyacrylamide gel. The observed bands a, b and bb are marked on the right. The bound and free DNA forms are indicated on the left. An asterisk indicates the 5′-end-labeled strand.

    Techniques Used: Binding Assay, Concentration Assay, Labeling

    Glycosylase stimulation by Ape1 and H309N. The glycosylase reactions were carried out at 37°C for 30 min with 0.2 nM A/GO substrate and increasing concentrations of Myh in the presence of 50 nM wild-type Ape1 (wt Ape1), 50 nM catalytic mutant H309N (H309N mutant), 50 nM BSA or buffer. Then each reaction mixture, with alkaline treatment, was analyzed on a 15% denaturing polyacrylamide gel. A plot of the fraction of glycosylase product versus [Myh] is shown.
    Figure Legend Snippet: Glycosylase stimulation by Ape1 and H309N. The glycosylase reactions were carried out at 37°C for 30 min with 0.2 nM A/GO substrate and increasing concentrations of Myh in the presence of 50 nM wild-type Ape1 (wt Ape1), 50 nM catalytic mutant H309N (H309N mutant), 50 nM BSA or buffer. Then each reaction mixture, with alkaline treatment, was analyzed on a 15% denaturing polyacrylamide gel. A plot of the fraction of glycosylase product versus [Myh] is shown.

    Techniques Used: Mutagenesis

    Effect of Ape1 concentration on glycosylase stimulation. The glycosylase reactions were carried out at 37°C for 30 min with 0.2 nM A/GO and 0.5 nM Myh in the presence of increasing amounts of Ape1. 5 nM Myh is also included. Then each reaction mixture, with alkaline treatment, was analyzed on a 15% denaturing polyacrylamide gel. The intact 96mer DNA (Intact) and cleaved 60mer product (Cleaved product) are indicated on the left. An asterisk indicates the 5′-end-labeled strand.
    Figure Legend Snippet: Effect of Ape1 concentration on glycosylase stimulation. The glycosylase reactions were carried out at 37°C for 30 min with 0.2 nM A/GO and 0.5 nM Myh in the presence of increasing amounts of Ape1. 5 nM Myh is also included. Then each reaction mixture, with alkaline treatment, was analyzed on a 15% denaturing polyacrylamide gel. The intact 96mer DNA (Intact) and cleaved 60mer product (Cleaved product) are indicated on the left. An asterisk indicates the 5′-end-labeled strand.

    Techniques Used: Concentration Assay, Labeling

    Myh–DNA complex formation. The glycosylase reaction was carried out at 37°C for 30 min with 0.2 nM substrate DNA and 5 nM Myh (saturating concentration) in the presence of 5 nM Ape1. Myh–DNA complexes were analyzed by EMSA on an 8% native polyacrylamide gel. The observed bands a, b and bb are marked on the right. The bound and free DNA forms are indicated on the left. An asterisk indicates the 5′-end-labeled strand.
    Figure Legend Snippet: Myh–DNA complex formation. The glycosylase reaction was carried out at 37°C for 30 min with 0.2 nM substrate DNA and 5 nM Myh (saturating concentration) in the presence of 5 nM Ape1. Myh–DNA complexes were analyzed by EMSA on an 8% native polyacrylamide gel. The observed bands a, b and bb are marked on the right. The bound and free DNA forms are indicated on the left. An asterisk indicates the 5′-end-labeled strand.

    Techniques Used: Concentration Assay, Labeling

    31) Product Images from "Incidence and persistence of 8-oxo-7,8-dihydroguanine within a hairpin intermediate exacerbates a toxic oxidation cycle associated with trinucleotide repeat expansion"

    Article Title: Incidence and persistence of 8-oxo-7,8-dihydroguanine within a hairpin intermediate exacerbates a toxic oxidation cycle associated with trinucleotide repeat expansion

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2011.06.003

    Data obtained under multiple-turnover conditions for determination of k 3 . Shown is a graph of concentration of product as a function of time for (A) Mixed-DUP, (B) Loop-DUP, (C) Stem-DUP, and (D) Stem-HP. Conditions were 50 nM DNA, 5 nM hOGG1 or 5 nM hOGG1/50 nM APE1 in 20 mM Tris-HCl, 70 mM NaCl, 2 mM MgCl 2 , 100 µg/mL BSA, pH 7.6.
    Figure Legend Snippet: Data obtained under multiple-turnover conditions for determination of k 3 . Shown is a graph of concentration of product as a function of time for (A) Mixed-DUP, (B) Loop-DUP, (C) Stem-DUP, and (D) Stem-HP. Conditions were 50 nM DNA, 5 nM hOGG1 or 5 nM hOGG1/50 nM APE1 in 20 mM Tris-HCl, 70 mM NaCl, 2 mM MgCl 2 , 100 µg/mL BSA, pH 7.6.

    Techniques Used: Concentration Assay

    32) Product Images from "Incidence and persistence of 8-oxo-7,8-dihydroguanine within a hairpin intermediate exacerbates a toxic oxidation cycle associated with trinucleotide repeat expansion"

    Article Title: Incidence and persistence of 8-oxo-7,8-dihydroguanine within a hairpin intermediate exacerbates a toxic oxidation cycle associated with trinucleotide repeat expansion

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2011.06.003

    Data obtained under multiple-turnover conditions for determination of k 3 . Shown is a graph of concentration of product as a function of time for (A) Mixed-DUP, (B) Loop-DUP, (C) Stem-DUP, and (D) Stem-HP. Conditions were 50 nM DNA, 5 nM hOGG1 or 5 nM hOGG1/50 nM APE1 in 20 mM Tris-HCl, 70 mM NaCl, 2 mM MgCl 2 , 100 µg/mL BSA, pH 7.6.
    Figure Legend Snippet: Data obtained under multiple-turnover conditions for determination of k 3 . Shown is a graph of concentration of product as a function of time for (A) Mixed-DUP, (B) Loop-DUP, (C) Stem-DUP, and (D) Stem-HP. Conditions were 50 nM DNA, 5 nM hOGG1 or 5 nM hOGG1/50 nM APE1 in 20 mM Tris-HCl, 70 mM NaCl, 2 mM MgCl 2 , 100 µg/mL BSA, pH 7.6.

    Techniques Used: Concentration Assay

    33) Product Images from "Incidence and persistence of 8-oxo-7,8-dihydroguanine within a hairpin intermediate exacerbates a toxic oxidation cycle associated with trinucleotide repeat expansion"

    Article Title: Incidence and persistence of 8-oxo-7,8-dihydroguanine within a hairpin intermediate exacerbates a toxic oxidation cycle associated with trinucleotide repeat expansion

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2011.06.003

    Data obtained under multiple-turnover conditions for determination of k 3 . Shown is a graph of concentration of product as a function of time for (A) Mixed-DUP, (B) Loop-DUP, (C) Stem-DUP, and (D) Stem-HP. Conditions were 50 nM DNA, 5 nM hOGG1 or 5 nM hOGG1/50 nM APE1 in 20 mM Tris-HCl, 70 mM NaCl, 2 mM MgCl 2 , 100 µg/mL BSA, pH 7.6.
    Figure Legend Snippet: Data obtained under multiple-turnover conditions for determination of k 3 . Shown is a graph of concentration of product as a function of time for (A) Mixed-DUP, (B) Loop-DUP, (C) Stem-DUP, and (D) Stem-HP. Conditions were 50 nM DNA, 5 nM hOGG1 or 5 nM hOGG1/50 nM APE1 in 20 mM Tris-HCl, 70 mM NaCl, 2 mM MgCl 2 , 100 µg/mL BSA, pH 7.6.

    Techniques Used: Concentration Assay

    34) Product Images from "The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes *"

    Article Title: The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.441444

    Assessment of the removal of rotationally and translationally positioned uracils by UDG and APE1. A, NCPs containing a single uracil at different sites were incubated with UDG and APE1. Open symbols represent in uracils as follows: red square , NCP-UI
    Figure Legend Snippet: Assessment of the removal of rotationally and translationally positioned uracils by UDG and APE1. A, NCPs containing a single uracil at different sites were incubated with UDG and APE1. Open symbols represent in uracils as follows: red square , NCP-UI

    Techniques Used: Incubation

    UDG and APE1 Digestion
    Figure Legend Snippet: UDG and APE1 Digestion

    Techniques Used:

    Polymerase β extension activity in NCPs near the dyad. A, representative gels for NCP-gO (+10) and NCP-gI (+4) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (+10) and NCP-gI (+4) were incubated with pol β and APE1
    Figure Legend Snippet: Polymerase β extension activity in NCPs near the dyad. A, representative gels for NCP-gO (+10) and NCP-gI (+4) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (+10) and NCP-gI (+4) were incubated with pol β and APE1

    Techniques Used: Activity Assay, Incubation

    Polymerase β extension activity in NCPs near DNA ends. A, representative gels for NCP-gO (−35) and NCP-gI (−49) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (−35) and NCP-gI (−49) were incubated
    Figure Legend Snippet: Polymerase β extension activity in NCPs near DNA ends. A, representative gels for NCP-gO (−35) and NCP-gI (−49) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (−35) and NCP-gI (−49) were incubated

    Techniques Used: Activity Assay, Incubation

    UDG and APE1 Digestion
    Figure Legend Snippet: UDG and APE1 Digestion

    Techniques Used:

    35) Product Images from "The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes *"

    Article Title: The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.441444

    Assessment of the removal of rotationally and translationally positioned uracils by UDG and APE1. A, NCPs containing a single uracil at different sites were incubated with UDG and APE1. Open symbols represent in uracils as follows: red square , NCP-UI
    Figure Legend Snippet: Assessment of the removal of rotationally and translationally positioned uracils by UDG and APE1. A, NCPs containing a single uracil at different sites were incubated with UDG and APE1. Open symbols represent in uracils as follows: red square , NCP-UI

    Techniques Used: Incubation

    UDG and APE1 Digestion
    Figure Legend Snippet: UDG and APE1 Digestion

    Techniques Used:

    Polymerase β extension activity in NCPs near the dyad. A, representative gels for NCP-gO (+10) and NCP-gI (+4) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (+10) and NCP-gI (+4) were incubated with pol β and APE1
    Figure Legend Snippet: Polymerase β extension activity in NCPs near the dyad. A, representative gels for NCP-gO (+10) and NCP-gI (+4) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (+10) and NCP-gI (+4) were incubated with pol β and APE1

    Techniques Used: Activity Assay, Incubation

    Polymerase β extension activity in NCPs near DNA ends. A, representative gels for NCP-gO (−35) and NCP-gI (−49) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (−35) and NCP-gI (−49) were incubated
    Figure Legend Snippet: Polymerase β extension activity in NCPs near DNA ends. A, representative gels for NCP-gO (−35) and NCP-gI (−49) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (−35) and NCP-gI (−49) were incubated

    Techniques Used: Activity Assay, Incubation

    UDG and APE1 Digestion
    Figure Legend Snippet: UDG and APE1 Digestion

    Techniques Used:

    36) Product Images from "The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes *"

    Article Title: The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.441444

    Assessment of the removal of rotationally and translationally positioned uracils by UDG and APE1. A, NCPs containing a single uracil at different sites were incubated with UDG and APE1. Open symbols represent in uracils as follows: red square , NCP-UI
    Figure Legend Snippet: Assessment of the removal of rotationally and translationally positioned uracils by UDG and APE1. A, NCPs containing a single uracil at different sites were incubated with UDG and APE1. Open symbols represent in uracils as follows: red square , NCP-UI

    Techniques Used: Incubation

    UDG and APE1 Digestion
    Figure Legend Snippet: UDG and APE1 Digestion

    Techniques Used:

    Polymerase β extension activity in NCPs near the dyad. A, representative gels for NCP-gO (+10) and NCP-gI (+4) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (+10) and NCP-gI (+4) were incubated with pol β and APE1
    Figure Legend Snippet: Polymerase β extension activity in NCPs near the dyad. A, representative gels for NCP-gO (+10) and NCP-gI (+4) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (+10) and NCP-gI (+4) were incubated with pol β and APE1

    Techniques Used: Activity Assay, Incubation

    Polymerase β extension activity in NCPs near DNA ends. A, representative gels for NCP-gO (−35) and NCP-gI (−49) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (−35) and NCP-gI (−49) were incubated
    Figure Legend Snippet: Polymerase β extension activity in NCPs near DNA ends. A, representative gels for NCP-gO (−35) and NCP-gI (−49) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (−35) and NCP-gI (−49) were incubated

    Techniques Used: Activity Assay, Incubation

    UDG and APE1 Digestion
    Figure Legend Snippet: UDG and APE1 Digestion

    Techniques Used:

    37) Product Images from "The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes *"

    Article Title: The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.441444

    Assessment of the removal of rotationally and translationally positioned uracils by UDG and APE1. A, NCPs containing a single uracil at different sites were incubated with UDG and APE1. Open symbols represent in uracils as follows: red square , NCP-UI
    Figure Legend Snippet: Assessment of the removal of rotationally and translationally positioned uracils by UDG and APE1. A, NCPs containing a single uracil at different sites were incubated with UDG and APE1. Open symbols represent in uracils as follows: red square , NCP-UI

    Techniques Used: Incubation

    UDG and APE1 Digestion
    Figure Legend Snippet: UDG and APE1 Digestion

    Techniques Used:

    Polymerase β extension activity in NCPs near the dyad. A, representative gels for NCP-gO (+10) and NCP-gI (+4) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (+10) and NCP-gI (+4) were incubated with pol β and APE1
    Figure Legend Snippet: Polymerase β extension activity in NCPs near the dyad. A, representative gels for NCP-gO (+10) and NCP-gI (+4) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (+10) and NCP-gI (+4) were incubated with pol β and APE1

    Techniques Used: Activity Assay, Incubation

    Polymerase β extension activity in NCPs near DNA ends. A, representative gels for NCP-gO (−35) and NCP-gI (−49) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (−35) and NCP-gI (−49) were incubated
    Figure Legend Snippet: Polymerase β extension activity in NCPs near DNA ends. A, representative gels for NCP-gO (−35) and NCP-gI (−49) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (−35) and NCP-gI (−49) were incubated

    Techniques Used: Activity Assay, Incubation

    UDG and APE1 Digestion
    Figure Legend Snippet: UDG and APE1 Digestion

    Techniques Used:

    38) Product Images from "Development of APE1 enzymatic DNA repair assays: low APE1 activity is associated with increase lung cancer risk"

    Article Title: Development of APE1 enzymatic DNA repair assays: low APE1 activity is associated with increase lung cancer risk

    Journal: Carcinogenesis

    doi: 10.1093/carcin/bgv082

    Optimizations of the radioactivity-based APE1 DNA repair assay. ( A ) Optimization of the preparation of protein extracts. Protein extracts were prepared under various conditions and assayed for APE1 activity. Lanes 1–3 Freeze-Thaw extraction; Lanes 4–7 extraction with NP40; Lanes 8–11 extraction with Triton; Lanes 12–13 Extraction by sonication; Lanes 14–15 Extraction by Syringe. ( B ) Effects of buffers and pH on APE1 activity. APE1 enzyme activity is presented relative to the activity in Tris pH 7.8 (set as 100%). Lanes 1–6 Tris buffer; Lanes 7–11 Phosphate buffer; Lanes 12–16 MOPS buffer; Lanes 17–20 Tricine buffer. ( C ) Effect of different salt concentrations on APE1 activity. APE1 enzyme activity is presented relative to the activity in 50mM KCl (set as 100%) Closed circles, KCl; Closed squares, NaCl. ( D ) Effect of MgCl 2 concentrations on APE1 activity. APE1 enzyme activity is presented relative to the activity in 9mM MgCl 2 (set as 100%). ( E ) Time course of APE1 DNA repair activity in protein extracts prepared from peripheral blood mononuclear cells. Closed squares, reaction under optimized conditions; Open squares, reaction before optimization; Closed circles, control DNA without the abasic site. ( F ) Relative frequency plots for APE1 activities were determined in 99 case patients (continuous line) and 99 matched controls subjects (dashed line). The relative frequencies as percent were plotted using GraphPad Prism version 5.00, with bin width of 100 units that was automatically chosen by the software. The relative frequency plots were smoothed by two neighbors on each size, zero order of polynomial smoothing. Case patients exhibit a shift to lower values of APE1 enzyme activity.
    Figure Legend Snippet: Optimizations of the radioactivity-based APE1 DNA repair assay. ( A ) Optimization of the preparation of protein extracts. Protein extracts were prepared under various conditions and assayed for APE1 activity. Lanes 1–3 Freeze-Thaw extraction; Lanes 4–7 extraction with NP40; Lanes 8–11 extraction with Triton; Lanes 12–13 Extraction by sonication; Lanes 14–15 Extraction by Syringe. ( B ) Effects of buffers and pH on APE1 activity. APE1 enzyme activity is presented relative to the activity in Tris pH 7.8 (set as 100%). Lanes 1–6 Tris buffer; Lanes 7–11 Phosphate buffer; Lanes 12–16 MOPS buffer; Lanes 17–20 Tricine buffer. ( C ) Effect of different salt concentrations on APE1 activity. APE1 enzyme activity is presented relative to the activity in 50mM KCl (set as 100%) Closed circles, KCl; Closed squares, NaCl. ( D ) Effect of MgCl 2 concentrations on APE1 activity. APE1 enzyme activity is presented relative to the activity in 9mM MgCl 2 (set as 100%). ( E ) Time course of APE1 DNA repair activity in protein extracts prepared from peripheral blood mononuclear cells. Closed squares, reaction under optimized conditions; Open squares, reaction before optimization; Closed circles, control DNA without the abasic site. ( F ) Relative frequency plots for APE1 activities were determined in 99 case patients (continuous line) and 99 matched controls subjects (dashed line). The relative frequencies as percent were plotted using GraphPad Prism version 5.00, with bin width of 100 units that was automatically chosen by the software. The relative frequency plots were smoothed by two neighbors on each size, zero order of polynomial smoothing. Case patients exhibit a shift to lower values of APE1 enzyme activity.

    Techniques Used: Radioactivity, Activity Assay, Sonication, Software

    APE1 DNA repair assay. ( A ) The structure of a furanyl abasic site. ( B ) Outline of the APE1 DNA repair assay. A radiolabeled synthetic short double-stranded DNA carrying a site-specific furanyl abasic site (marked by a circle) was incubated with a protein extract. The action of APE1 caused a nick at the lesion site, which enabled subsequent quantification of reaction products. ( C ) Inhibition of DNA repair activities by the APE1 inhibitor NICA. Closed squares, purified APE1 enzyme (0.1 unit per in the reaction); Open squares, APE1 activity in PBMC extract (0.025ng/µl protein extract in the reaction); Open triangles, OGG1 activity in PBMC extract (0.4 µg/µl protein extract in reaction).
    Figure Legend Snippet: APE1 DNA repair assay. ( A ) The structure of a furanyl abasic site. ( B ) Outline of the APE1 DNA repair assay. A radiolabeled synthetic short double-stranded DNA carrying a site-specific furanyl abasic site (marked by a circle) was incubated with a protein extract. The action of APE1 caused a nick at the lesion site, which enabled subsequent quantification of reaction products. ( C ) Inhibition of DNA repair activities by the APE1 inhibitor NICA. Closed squares, purified APE1 enzyme (0.1 unit per in the reaction); Open squares, APE1 activity in PBMC extract (0.025ng/µl protein extract in the reaction); Open triangles, OGG1 activity in PBMC extract (0.4 µg/µl protein extract in reaction).

    Techniques Used: Incubation, Inhibition, Purification, Activity Assay

    Fluorescence-based APE1 DNA repair assay. ( A ) Example of a fluorescent plot of the APE1 reaction products analyzed by capillary gel electrophoresis, using the ABI3130XL genetic analyzer and the GeneMapper software. ( B and C ) Time course and protein extract titration, respectively, of APE1 DNA repair activity under optimized reaction conditions in protein extracts prepared from peripheral blood mononuclear cells. Quantification was done by quantifying fluorescent plots such as the one presented in A. Closed circles, substrate with an abasic site; Open circles, control DNAs without the lesion. ( D and E ) Correlation between the radioactivity-based APE1 assay (APE1-P) and the fluorescence-based APE1 assay (APE1-F) in control subjects (closed squares; D) and case patients (closed circles; E).
    Figure Legend Snippet: Fluorescence-based APE1 DNA repair assay. ( A ) Example of a fluorescent plot of the APE1 reaction products analyzed by capillary gel electrophoresis, using the ABI3130XL genetic analyzer and the GeneMapper software. ( B and C ) Time course and protein extract titration, respectively, of APE1 DNA repair activity under optimized reaction conditions in protein extracts prepared from peripheral blood mononuclear cells. Quantification was done by quantifying fluorescent plots such as the one presented in A. Closed circles, substrate with an abasic site; Open circles, control DNAs without the lesion. ( D and E ) Correlation between the radioactivity-based APE1 assay (APE1-P) and the fluorescence-based APE1 assay (APE1-F) in control subjects (closed squares; D) and case patients (closed circles; E).

    Techniques Used: Fluorescence, Nucleic Acid Electrophoresis, Software, Titration, Activity Assay, Radioactivity

    39) Product Images from "Alzheimer’s Disease Associated Polymorphisms in Human OGG1 Alter Catalytic Activity and Sensitize Cells to DNA Damage"

    Article Title: Alzheimer’s Disease Associated Polymorphisms in Human OGG1 Alter Catalytic Activity and Sensitize Cells to DNA Damage

    Journal: Free radical biology & medicine

    doi: 10.1016/j.freeradbiomed.2013.05.010

    Decreased AP lyase activity and differential stimulation of polymorphic OGG1s by APE1 (A) OGG1 recombinant proteins (64nM) were incubated at 37°C for 15 minutes in the presence (+) or absence (−) of 45pM APE1, along with a 5′-end-labeled oligonucleotide duplex containing an 8-oxoG lesion. The cleavage products were analyzed on a 15% polyacrylamide gel containing 7M Urea and imaged on a phosphorimager. A representative experiment is shown. (B) The histogram represents the mean ± S.E.M. from five independent experiments. The incision value was calculated by taking the amount of cleaved substrate (lower band) normalized to the amount of cleaved + uncleaved substrate (lower + upper bands) . *p
    Figure Legend Snippet: Decreased AP lyase activity and differential stimulation of polymorphic OGG1s by APE1 (A) OGG1 recombinant proteins (64nM) were incubated at 37°C for 15 minutes in the presence (+) or absence (−) of 45pM APE1, along with a 5′-end-labeled oligonucleotide duplex containing an 8-oxoG lesion. The cleavage products were analyzed on a 15% polyacrylamide gel containing 7M Urea and imaged on a phosphorimager. A representative experiment is shown. (B) The histogram represents the mean ± S.E.M. from five independent experiments. The incision value was calculated by taking the amount of cleaved substrate (lower band) normalized to the amount of cleaved + uncleaved substrate (lower + upper bands) . *p

    Techniques Used: Activity Assay, Recombinant, Incubation, Labeling

    40) Product Images from "Influence of Oxidized Purine Processing on Strand Directionality of Mismatch Repair *"

    Article Title: Influence of Oxidized Purine Processing on Strand Directionality of Mismatch Repair *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.629907

    MYH is active in HCT116 extracts and addresses G O /A but not A O /G or A O /C mispairs. A , nicking assay. The closed-circular homoduplex or the G O /A, A O /G, or A O /C substrates were incubated with recombinant, purified MYH-GST and APE1 for the indicated times
    Figure Legend Snippet: MYH is active in HCT116 extracts and addresses G O /A but not A O /G or A O /C mispairs. A , nicking assay. The closed-circular homoduplex or the G O /A, A O /G, or A O /C substrates were incubated with recombinant, purified MYH-GST and APE1 for the indicated times

    Techniques Used: Incubation, Recombinant, Purification

    Related Articles

    In Vitro:

    Article Title: Deletion of Individual Ku Subunits in Mice Causes an NHEJ-Independent Phenotype Potentially by Altering Apurinic/Apyrimidinic Site Repair
    Article Snippet: .. Standard reaction mixtures (100 µl) for the molecular beacon assay contained 20 mM Tris-acetate, pH 7.9, 10 mM Magnesium acetate, 50 mM Potassium acetate, 1 mM dithiothreitol, 250 nM AP DNA duplex and in vitro translation samples (40 µg) and APE1 (10 u) were prepared on ice and then measured at 37°C, every 10 sec. for 10 min. ..

    Size-exclusion Chromatography:

    Article Title: Deletion of Individual Ku Subunits in Mice Causes an NHEJ-Independent Phenotype Potentially by Altering Apurinic/Apyrimidinic Site Repair
    Article Snippet: .. Standard reaction mixtures (100 µl) for the molecular beacon assay contained 20 mM Tris-acetate, pH 7.9, 10 mM Magnesium acetate, 50 mM Potassium acetate, 1 mM dithiothreitol, 250 nM AP DNA duplex and in vitro translation samples (40 µg) and APE1 (10 u) were prepared on ice and then measured at 37°C, every 10 sec. for 10 min. ..

    Incubation:

    Article Title: Influence of Oxidized Purine Processing on Strand Directionality of Mismatch Repair *
    Article Snippet: .. We therefore asked whether incubation of the supercoiled GO /A substrate with purified, recombinant MYH-GST and APE1 gave rise to nicked circular molecules. .. As shown in A ( lanes 3–6 ), the GO /A substrate was efficiently converted to the open-circular form upon incubation of the plasmid with the recombinant proteins.

    Purification:

    Article Title: CUX1 stimulates APE1 enzymatic activity and increases the resistance of glioblastoma cells to the mono-alkylating agent temozolomide
    Article Snippet: .. Endonuclease reactions with bacterially purified proteins were conducted using 0.08 nM APE1 (New England Biolabs) and 50 nM bovine serum albumin (BSA) or the indicated proteins unless otherwise indicated and 1 pmol of 32 P-radiolabeled double-stranded oligonucleotides containing a tetrahydrofuran site or an abasic site generated by uracil-DNA glycosylase (UDG)/MPG and performed as described. ..

    Article Title: Influence of Oxidized Purine Processing on Strand Directionality of Mismatch Repair *
    Article Snippet: .. We therefore asked whether incubation of the supercoiled GO /A substrate with purified, recombinant MYH-GST and APE1 gave rise to nicked circular molecules. .. As shown in A ( lanes 3–6 ), the GO /A substrate was efficiently converted to the open-circular form upon incubation of the plasmid with the recombinant proteins.

    Generated:

    Article Title: CUX1 stimulates APE1 enzymatic activity and increases the resistance of glioblastoma cells to the mono-alkylating agent temozolomide
    Article Snippet: .. Endonuclease reactions with bacterially purified proteins were conducted using 0.08 nM APE1 (New England Biolabs) and 50 nM bovine serum albumin (BSA) or the indicated proteins unless otherwise indicated and 1 pmol of 32 P-radiolabeled double-stranded oligonucleotides containing a tetrahydrofuran site or an abasic site generated by uracil-DNA glycosylase (UDG)/MPG and performed as described. ..

    other:

    Article Title: Alzheimer’s Disease Associated Polymorphisms in Human OGG1 Alter Catalytic Activity and Sensitize Cells to DNA Damage
    Article Snippet: In contrast to A53T, the A288V polymorphism was sufficiently activated by APE1.

    Article Title: Oxidative modification of guanine in a potential Z-DNA-forming sequence of a gene promoter impacts gene expression
    Article Snippet: Studies to understand whether APE1 could bind F in the loop of a hairpin were not pursued; however, previous reports have found APE1 binds AP in loops while poorly cleaving the strand.

    Activity Assay:

    Article Title: Alzheimer’s Disease Associated Polymorphisms in Human OGG1 Alter Catalytic Activity and Sensitize Cells to DNA Damage
    Article Snippet: .. Although APE1 stimulated the activity of the A53T protein, the level of incision was still significantly lower than WT stimulated by APE1 (p < 0.01). .. APE1 stimulated the A288V polymorphic protein above APE1 stimulation of WT but this increase was not statistically significant.

    Article Title: Alzheimer’s Disease Associated Polymorphisms in Human OGG1 Alter Catalytic Activity and Sensitize Cells to DNA Damage
    Article Snippet: .. One well established step of BER occurs when APE1 binds directly to OGG1 and stimulates its activity [ – , ]. .. We found that APE1 significantly stimulated the incision ability of WT OGG1.

    Recombinant:

    Article Title: Influence of Oxidized Purine Processing on Strand Directionality of Mismatch Repair *
    Article Snippet: .. We therefore asked whether incubation of the supercoiled GO /A substrate with purified, recombinant MYH-GST and APE1 gave rise to nicked circular molecules. .. As shown in A ( lanes 3–6 ), the GO /A substrate was efficiently converted to the open-circular form upon incubation of the plasmid with the recombinant proteins.

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    New England Biolabs ape1
    Decreased AP lyase activity and differential stimulation of polymorphic OGG1s by <t>APE1</t> (A) OGG1 recombinant proteins (64nM) were incubated at 37°C for 15 minutes in the presence (+) or absence (−) of 45pM APE1, along with a 5′-end-labeled oligonucleotide duplex containing an 8-oxoG lesion. The cleavage products were analyzed on a 15% polyacrylamide gel containing 7M Urea and imaged on a phosphorimager. A representative experiment is shown. (B) The histogram represents the mean ± S.E.M. from five independent experiments. The incision value was calculated by taking the amount of cleaved substrate (lower band) normalized to the amount of cleaved + uncleaved substrate (lower + upper bands) . *p
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    Decreased AP lyase activity and differential stimulation of polymorphic OGG1s by APE1 (A) OGG1 recombinant proteins (64nM) were incubated at 37°C for 15 minutes in the presence (+) or absence (−) of 45pM APE1, along with a 5′-end-labeled oligonucleotide duplex containing an 8-oxoG lesion. The cleavage products were analyzed on a 15% polyacrylamide gel containing 7M Urea and imaged on a phosphorimager. A representative experiment is shown. (B) The histogram represents the mean ± S.E.M. from five independent experiments. The incision value was calculated by taking the amount of cleaved substrate (lower band) normalized to the amount of cleaved + uncleaved substrate (lower + upper bands) . *p

    Journal: Free radical biology & medicine

    Article Title: Alzheimer’s Disease Associated Polymorphisms in Human OGG1 Alter Catalytic Activity and Sensitize Cells to DNA Damage

    doi: 10.1016/j.freeradbiomed.2013.05.010

    Figure Lengend Snippet: Decreased AP lyase activity and differential stimulation of polymorphic OGG1s by APE1 (A) OGG1 recombinant proteins (64nM) were incubated at 37°C for 15 minutes in the presence (+) or absence (−) of 45pM APE1, along with a 5′-end-labeled oligonucleotide duplex containing an 8-oxoG lesion. The cleavage products were analyzed on a 15% polyacrylamide gel containing 7M Urea and imaged on a phosphorimager. A representative experiment is shown. (B) The histogram represents the mean ± S.E.M. from five independent experiments. The incision value was calculated by taking the amount of cleaved substrate (lower band) normalized to the amount of cleaved + uncleaved substrate (lower + upper bands) . *p

    Article Snippet: Although APE1 stimulated the activity of the A53T protein, the level of incision was still significantly lower than WT stimulated by APE1 (p < 0.01).

    Techniques: Activity Assay, Recombinant, Incubation, Labeling