Journal: Molecular cell

Article Title: APE1-dependent base excision repair of DNA photodimers in human cells.

doi: 10.1016/j.molcel.2023.09.013

Figure Lengend Snippet: Figure 4. NER-independent repair of 6-4PP requires APE1 (A) Levels of XRCC1, APE1, and XPA protein levels in the indicated gene-edited RPE-1 cell clines, measured by western blotting. a-Tubulin was employed as a loading control. (B) ADP-ribose levels in XRCC1/ and XRCC1//APE1/ RPE-1 cells in G1 phase 60 min after MMS treatment (0.05 mg/mL). ADP-ribose levels are plotted relative to those in untreated XRCC1/ cells and data are the mean (± SEM) of three independent experiments. Symbols show individual data points. Statistically significant differences were determined by one-way ANOVA with Tukey’s post hoc multiple comparisons test (****p < 0.0001). (C) ADP-ribose levels in XRCC1/ and XRCC1//APE1/ RPE-1 cells in G1 phase 60 min after UVC irradiation (40 J/m2). Data and statistics as in (B). (***p < 0.001). (D) ADP-ribose levels in wild-type (WT) and the indicated gene-edited RPE-1 cells in G1 phase 60 min after UVC irradiation (40 J/m2) and incubation with or without PARGi for the final 30 min to prevent poly(ADP-ribose) degradation. Data are plotted relative to untreated wild-type cells and are mean (± SEM) of three inde- pendent experiments. Symbols show individual data points. Statistically significant differences were determined by one-way ANOVA with Tukey’s post hoc multiple comparisons test (*p < 0.05; **p < 0.01; ****p < 0.0001). (E) Levels of 6-4PP in genomic DNA purified from growth-arrested wild-type (WT) and the indicated mutant RPE-1 cells at the indicated times following UVC irradiation (15 J/m2). Data are the mean (± SEM) of 3–4 independent experiments, and statistical analysis was by mixed effects analysis with Sidak’s multiple comparisons test. See also Figure S4.

Article Snippet: The radiolabelled oligonucleotide duplexes were incubated with 2 pmol recombinant human APE1 and/or 4 pmol of recombinant NTH1 (OriGene) or 10 units of T4-Endonuclease V (NEB) for 60minutes at 37 C in reaction buffer (100mMKCl, 20mMTris, pH 7.5, 10mMMgCl2, 0.5mMDTT, 0.25%polyvinyl alcohol) and the reactions stopped and fractionated by denaturing PAGE as described above.

Techniques: Western Blot, Control, Irradiation, Incubation, Mutagenesis

Journal: Biomedicines

Article Title: Elevated Plasma Apurinic/Apyrimidinic Endonuclease 1/Redox Effector Factor-1 Levels in Refractory Kawasaki Disease

doi: 10.3390/biomedicines10010190

Figure Lengend Snippet: Laboratory data.

Article Snippet: Secreted APE1/Ref-1 levels in plasma (ng/mL) were calculated against a standard curve generated using recombinant human APE1/Ref-1 protein (MediRedox).

Techniques:

Journal: DNA repair

Article Title: The splicing component ISY1 regulates APE1 in base excision repair

doi: 10.1016/j.dnarep.2019.102769

Figure Lengend Snippet: ISY1 protein levels in different cell lines were assessed by western blot analysis. Panel a shows ISY1 and APE1 protein levels in HEK293, A549, H157, HCT, HCT-APE knock-down, MCF7, BPH, LnCap, PC3, U2OS, and U87MG cells. Panel b shows quantitation of ISY1 and APE1 after normalization with α-tubulin levels in HEK293, A549, H157, HCT116, HCT-APE1 knock-down, MCF7, BPH, LnCap, PC3, U2OS, and U87MG cells. Panel c shows the effect of ISY1 and APE1 depletion in HCT116 cells on the level of other BER proteins. Panel d shows the effect of varying concentration of MMS (0–1000 μM) treatment on the expression level of ISY1 and

Article Snippet: Human recombinant APE1 was purchased from New England Biolab (NEB, Ipswich, MA, USA) and human recombinant ISY1 protein was purchased from Abnova (Walnut, CA, USA).

Techniques: Western Blot, Quantitation Assay, Concentration Assay, Expressing

Journal: DNA repair

Article Title: The splicing component ISY1 regulates APE1 in base excision repair

doi: 10.1016/j.dnarep.2019.102769

Figure Lengend Snippet: Panel a show the experimental protocol. Panel b shows the effect of varying concentrations of ISY1 protein on APE1’s 5’−3’ endonuclease activity on AP-containing double stranded DNA. The top 32P-labeled strand harbors a 3-hydroxy-2-hydroxymethyltetrahydrofuran moiety (F). The incubation time was 45 min. Panel c shows the effect of time of incubation with limiting amounts of ISY1 and APE1 on the cleavage of 63-mer 32P-labeled F-DNA substrate. The time points were 15, 30, 45 and 60 min. Data are the mean ± SD of three independent experiments.

Article Snippet: Human recombinant APE1 was purchased from New England Biolab (NEB, Ipswich, MA, USA) and human recombinant ISY1 protein was purchased from Abnova (Walnut, CA, USA).

Techniques: Activity Assay, Labeling, Incubation

Journal: DNA repair

Article Title: The splicing component ISY1 regulates APE1 in base excision repair

doi: 10.1016/j.dnarep.2019.102769

Figure Lengend Snippet: Panel a is the schematic representation for assembly of the LP-BER system. Panel b shows the efficiency of ISY1/APE1 incision at an AP site and processing by down-stream BER enzymes. The incubation time was 60 min. Lane 1 represents 32P-63-mer F-DNA, lane 2 shows 23-mer incised product after excessive APE1 activity, and lane 3 shows displacement activity in the presence of POL β. Lane 5 shows Fen1 stimulated POL β activity, and lane 7 shows the repair of the 63-mer AP site DNA. Lanes 8–14 show a LP-BER reaction in the presence of limiting concentrations of APE1. Lanes 15–24 show the LP-BER reaction reconstituted in the presence of ISY1. Lower panel shows quantitation of uncleaved product representing unrepaired product in absence of DNA ligase 1 and repaired product in presence of DNA ligase 1. * lanes indicate reaction in presence of DNA ligase 1. Incubation time for the reaction was 60 min. Data are the mean ± SD of three independent experiments.

Article Snippet: Human recombinant APE1 was purchased from New England Biolab (NEB, Ipswich, MA, USA) and human recombinant ISY1 protein was purchased from Abnova (Walnut, CA, USA).

Techniques: Incubation, Activity Assay, Quantitation Assay

Journal: DNA repair

Article Title: The splicing component ISY1 regulates APE1 in base excision repair

doi: 10.1016/j.dnarep.2019.102769

Figure Lengend Snippet: Binding affinities of ISY1 and APE1 were measured by incubating with 32P-F-DNA. The reaction was assembled on ice and nucleoprotein complexes were resolved on a 5.5% non-denaturing polyacrylamide gel. Panel a, APE1 (10–50 ng) and ISY1 (25–100 ng) were tested for binding either the control or F-DNA. Lane 1, no protein; lanes 2–4, APE1 (10–25 ng); lanes 5–7, ISY1 (10–50 ng) were incubated with 32P-control or 32P-F DNA; lanes 8–10, APE1 (10–25 ng); lanes 11–13 ISY1 (25–100 ng). Panel 6b, supershifting of the APE1-F-DNA complex by anti-APE1 antibody (2 μg). Panel 6c, ISY1 enhances APE1 binding to AP-DNA. APE1 binding to AP site DNA was assembled on ice. Following the incubation of 32P-F-DNA with APE1 (10–50 ng) and ISY1 (25–100 ng) for 10 min, DNA:Protein complexes were resolved on a 5.5% non-denaturing polyacrylamide gel. The results in Panel 6c were quantified and plotted. Lane 1, no protein; lanes 2–4, APE1 (25–100 ng); lanes 5–7, ISY1 (25–100 ng) were incubated with 32P-labeled AP site DNA; lanes 8–10, 100 ng ISY1 and varying amount of APE1 (10–25 ng); lanes 11–13, APE1 (25 ng) and varying amount of ISY1 (25–100 ng) were incubated with 32P-labeled AP-DNA. Panel 6d shows quantitation of the binding of ISY1 and APE1 proteins to AP site DNA. Data are representative of three independent experiments. The arrows indicate the position of free and shifted bands. Data are the mean ± SD of three independent experiments. * P < 0.05 and ** P<0.005, significantly different than control.

Article Snippet: Human recombinant APE1 was purchased from New England Biolab (NEB, Ipswich, MA, USA) and human recombinant ISY1 protein was purchased from Abnova (Walnut, CA, USA).

Techniques: Binding Assay, Incubation, Labeling, Quantitation Assay

Journal: DNA repair

Article Title: The splicing component ISY1 regulates APE1 in base excision repair

doi: 10.1016/j.dnarep.2019.102769

Figure Lengend Snippet: ISY1 knock-down cells were transfected with Flag-APE1 wt plasmid. Cells were treated with the concentration of 75 and 100 μM of MMS and 5 &10 μM H2O2 for 24 h. After treatment, the medium was replaced with a fresh medium containing 10% (v/v) fetal bovine serum. Cells were allowed to grow till visible colonies are formed. Colonies were stained, counted and analyzed. panel a, colony assay after MMS treatment (representative picture); panel b and d, quantitation of colony assay; panel c, colony assay after H2O2 treatment (representative picture). Panel e, western analysis of ISY1 and APE proteins. β-Actin served as a loading control. Data are the mean ± SD. * P < 0.05, ** P<0.005 and *** P<0.001, significantly different than control.

Article Snippet: Human recombinant APE1 was purchased from New England Biolab (NEB, Ipswich, MA, USA) and human recombinant ISY1 protein was purchased from Abnova (Walnut, CA, USA).

Techniques: Transfection, Plasmid Preparation, Concentration Assay, Staining, Colony Assay, Quantitation Assay, Western Blot