Journal: bioRxiv

Article Title: Conditional deletion of human STN1 leads to telomere dysfunction and telomerase-dependent genome instability and proliferation defects

doi: 10.1101/2025.08.06.669015

Figure Lengend Snippet: (A) Western blot of STN1 levels in HeLa iCas9 sgSTN1 cells, as indicated. Actinin serves as a loading control. DOX=Doxycycline. (B) Growth curve analysis of STN1 KO and control cells. D=day after DOX addition. n=3 independent, biological replicates. (C-D) Flow cytometry analysis of STN1 KO cells. (C) Representative histograms of STN1 KO and control (-DOX) cells. Left, histograms of DNA content versus cell count. Right, DNA content versus EdU intensity. (D) Percentage of G2/M cells over time in STN1 KO cells. n=3 independent, biological replicates. Average values in the graphs indicate the mean and error bars denote ±s.e.m. P -values were calculated by a two-tailed, unpaired t -test.

Article Snippet: Primary: OBFC1 (STN1) (Novus, NBP2-01006), Actinin (Santa Cruz, sc17829), pH3 S10 (Cell Signaling, 9706), RPA32 (Abcam, ab16850), and γH2AX (Bethyl, A300-081A; Abcam, ab81299).

Techniques: Western Blot, Control, Flow Cytometry, Cell Counting, Two Tailed Test

Journal: bioRxiv

Article Title: Conditional deletion of human STN1 leads to telomere dysfunction and telomerase-dependent genome instability and proliferation defects

doi: 10.1101/2025.08.06.669015

Figure Lengend Snippet: (A) Representative image of RPA-γH2AX foci. STN1 KO image is from day 12 after DOX addition. (B-C) Analysis of foci in STN1 KO and control (-DOX) cells. Percentage of nuclei displaying 3 or more RPA (B) or RPA-γH2AX foci (C). n=3 independent, biological replicates. Average values in the graphs indicate the mean and error bars denote ±s.e.m. P -values were calculated by a two-tailed, unpaired t -test. DOX=Doxycycline. D=day after DOX addition.

Article Snippet: Primary: OBFC1 (STN1) (Novus, NBP2-01006), Actinin (Santa Cruz, sc17829), pH3 S10 (Cell Signaling, 9706), RPA32 (Abcam, ab16850), and γH2AX (Bethyl, A300-081A; Abcam, ab81299).

Techniques: Control, Two Tailed Test

Journal: bioRxiv

Article Title: Conditional deletion of human STN1 leads to telomere dysfunction and telomerase-dependent genome instability and proliferation defects

doi: 10.1101/2025.08.06.669015

Figure Lengend Snippet: (A) Western blot of STN1 and Flag-STN1 levels, as indicated. KO+Flag-STN1 denotes STN1 KO cells expressing Flag-STN1. Actinin serves as a loading control. (B) Percentage of nuclei displaying 3 or more RPA foci on day 15 after DOX addition. n=3 independent, biological replicates. (C) Growth curve analysis, as indicated. n=3 independent, biological replicates. (D) Percentage of G2/M cells determined by flow cytometry on day 15 after DOX addition. n=4 independent, biological replicates. Average values in the graphs indicate the mean and error bars denote ±s.e.m. P -values were calculated by a two-tailed, unpaired t -test. DOX=Doxycycline.

Article Snippet: Primary: OBFC1 (STN1) (Novus, NBP2-01006), Actinin (Santa Cruz, sc17829), pH3 S10 (Cell Signaling, 9706), RPA32 (Abcam, ab16850), and γH2AX (Bethyl, A300-081A; Abcam, ab81299).

Techniques: Western Blot, Expressing, Control, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: Conditional deletion of human STN1 leads to telomere dysfunction and telomerase-dependent genome instability and proliferation defects

doi: 10.1101/2025.08.06.669015

Figure Lengend Snippet: (A) Representative image of an anaphase bridge (indicated by the arrow) in STN1 KO cells. (B-C) Percentage of anaphase bridges in STN1 KO and control (-DOX) cells between days 5-15 (B) or with expression of exogenous Flag-STN1 on day 15 (C). n=≥3 independent, biological replicates. (D) Representative image of micronuclei (indicated by the arrows) in the STN1 KO cells. (E-F) Percentage of cells with micronuclei in STN1 KO and control cells between days 5-15 (E) or with expression of exogenous Flag-STN1 on day 15 (F). n=≥3 independent, biological replicates. Average values in the graphs indicate the mean and error bars denote ±s.e.m. P -values were calculated by a two-tailed, unpaired t -test. DOX=Doxycycline. D=day after DOX addition.

Article Snippet: Primary: OBFC1 (STN1) (Novus, NBP2-01006), Actinin (Santa Cruz, sc17829), pH3 S10 (Cell Signaling, 9706), RPA32 (Abcam, ab16850), and γH2AX (Bethyl, A300-081A; Abcam, ab81299).

Techniques: Control, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Conditional deletion of human STN1 leads to telomere dysfunction and telomerase-dependent genome instability and proliferation defects

doi: 10.1101/2025.08.06.669015

Figure Lengend Snippet: Cells were treated with a telomerase inhibitor (BIBR) or DMSO starting with the addition of DOX on day 0 to the sgSTN1 cells. The control cells were also treated with BIBR or DMSO across the same timeframe. (A-B) Percentage of nuclei with 3 or more RPA (A) or RPA-γH2AX (B) foci in STN1 KO or control cells with or without BIBR treatment. n=3 independent, biological replicates. (C-D) Percentage of micronuclei (C) or anaphase bridges (D) on day 15 after DOX addition with and without BIBR treatment. n=4 independent, biological replicates. (E) Growth curve analysis, as indicated. n=3 independent, biological replicates. Average values in the graphs indicate the mean and error bars denote ±s.e.m. P -values were calculated by a two-tailed, unpaired t -test. DOX=Doxycycline. D=day after DOX addition.

Article Snippet: Primary: OBFC1 (STN1) (Novus, NBP2-01006), Actinin (Santa Cruz, sc17829), pH3 S10 (Cell Signaling, 9706), RPA32 (Abcam, ab16850), and γH2AX (Bethyl, A300-081A; Abcam, ab81299).

Techniques: Control, Two Tailed Test

Journal: Journal of molecular biology

Article Title: Human CST Stimulates Base Excision Repair to Prevent the Accumulation of Oxidative DNA Damage

doi: 10.1016/j.jmb.2024.168672

Figure Lengend Snippet: (A) Representative images of proximity ligation assay (PLA) performed in HeLa cells with antibodies to STN1 or Pol β alone or in combination with and without H 2 O 2 treatment. Red, PLA foci; blue, DAPI. (B) Violin plot of PLA foci per nucleus. Results are representative of three independent, biological experiments. Bold dashed line represents the median, and dashed lines represent the first and third quartiles. (C) Proximity ligation assay (PLA) performed in HeLa cells with antibodies to STN1, OGG1, or in combination (STN1 + OGG1). n = 3 independent, biological experiments. Bold dashed line: median, dashed lines: first and third quartiles. (D) HEK293T cell extracts containing individually overexpressed CST components (either FLAG-CST, Myc-STN1 or FLAG-TEN1) that were either untreated or treated with 200 μM of H 2 O 2 were used to immunoprecipitate APE1, Pol β, FEN1 or LIGI and immunobloted with either anti-FLAG (for CTC1 and TEN1) or anti-Myc for STN1. Lysates were pre-treated with benzonase prior to coimmunoprecipitation. Proteins are identified in the Western blot analysis. (**** p < 0.0001).

Article Snippet: Primary antibodies: 1:100 mouse α-STN1 (Novus, NBP2-01006), 1:100 rabbit α-POLB (Abbexa, abx304879), 1:100 rabbit α-PolA1 (Bethyl Laboratories, A302-850A), 1:100 rabbit α-XRCC1 (Genetex, GTX111712), 1:100 mouse α-XRCC1 (Invitrogen, MA1-12640), 1:100 rabbit α-PARP1 (Proteintech, 6520-1-Ig), 1:100 rabbit α-OGG1 (Proteintech, 15125-1-AP).

Techniques: Proximity Ligation Assay, Western Blot

Journal: Journal of molecular biology

Article Title: Human CST Stimulates Base Excision Repair to Prevent the Accumulation of Oxidative DNA Damage

doi: 10.1016/j.jmb.2024.168672

Figure Lengend Snippet: (A) OGG1 (2 nM, lanes 2, 4–15; 50 nM, lane 3) lesion base removal and phosphodiester bone cleavage activity were measured on a random sequence DNA substrate (5 nM) containing an 8-oxo-guanine (8-oxo-G) residue in the presence of increasing concentrations of CTC1/STN1/TEN1 (250, 750, 1500 nM) or CST (5, 15, 60 nM) as denoted in the figure. The red asterisk shown on the substrate in the figure represents the location of the 32 P radiolabel and a G* represents an 8-oxo-G residue. Gel shown is representative of at least three independent experiments. (B) Graph of the fold increase in OGG1 cleavage activity for the lanes containing the highest concentration of the CST complex and individual subunits. The random substrate (U1:T1) was generated by annealing primers in a 1:2 ratio. (**** p < 0.0001).

Article Snippet: Primary antibodies: 1:100 mouse α-STN1 (Novus, NBP2-01006), 1:100 rabbit α-POLB (Abbexa, abx304879), 1:100 rabbit α-PolA1 (Bethyl Laboratories, A302-850A), 1:100 rabbit α-XRCC1 (Genetex, GTX111712), 1:100 mouse α-XRCC1 (Invitrogen, MA1-12640), 1:100 rabbit α-PARP1 (Proteintech, 6520-1-Ig), 1:100 rabbit α-OGG1 (Proteintech, 15125-1-AP).

Techniques: Activity Assay, Sequencing, Residue, Concentration Assay, Generated

Journal: Journal of molecular biology

Article Title: Human CST Stimulates Base Excision Repair to Prevent the Accumulation of Oxidative DNA Damage

doi: 10.1016/j.jmb.2024.168672

Figure Lengend Snippet: (A) APE1 (0.075 nM, lanes 2, 4–15; 100 nM, lane 3) cleavage activity was measured on a random sequence DNA substrate (5 nM) containing a THF residue in the presence of increasing concentrations of CTC1/STN1/TEN1 (250, 750, 1500 nM) or CST (5, 15, 60 nM) as denoted in the figure. The red asterisk shown on the substrate in the figure represents the location of the 32 P radiolabel and an Φ represents a THF residue. Gel shown is representative of at least three independent experiments. (B) Graph of the fold increase in APE1 cleavage activity for the lanes containing the highest concentration of the CST complex and individual subunits. (C) Graph of the fold increase in APE1 cleavage activity on a telomere substrate calculated as in (B). The error bars represent the standard error mean. The random substrate (U2:T1) and telomere substrate (U3:T2) was generated by annealing primers in a 1:2 ratio. (**** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05).

Article Snippet: Primary antibodies: 1:100 mouse α-STN1 (Novus, NBP2-01006), 1:100 rabbit α-POLB (Abbexa, abx304879), 1:100 rabbit α-PolA1 (Bethyl Laboratories, A302-850A), 1:100 rabbit α-XRCC1 (Genetex, GTX111712), 1:100 mouse α-XRCC1 (Invitrogen, MA1-12640), 1:100 rabbit α-PARP1 (Proteintech, 6520-1-Ig), 1:100 rabbit α-OGG1 (Proteintech, 15125-1-AP).

Techniques: Activity Assay, Sequencing, Residue, Concentration Assay, Generated

Journal: Journal of molecular biology

Article Title: Human CST Stimulates Base Excision Repair to Prevent the Accumulation of Oxidative DNA Damage

doi: 10.1016/j.jmb.2024.168672

Figure Lengend Snippet: Synthesis by DNA Pol β (2 nM) on a 1 nt gapped substrate (5 nM) containing an abasic site mimic (Φ denotes tetrahydrofuran [THF]) in the presence of increasing amounts of CTC1/STN1/TEN1 (100, 500, 1000 nM) or CST (5, 15, 30 nM) as denoted in the figure. The substrate was generated by annealing primers U4:T1: D1 in a 1:2:4 ratio. The red asterisk shown on the substrate represents the location of the 32 P radiolabel. Gel shown is representative of at least three independent experiments. A darker exposure of the gel shows the strand displacement synthesis products. (B) The trace at the right of the gel depicts the signal intensity of the gap-filling and strand-displacement products in the lanes containing the highest concentration of CTC1(red trace)/STN1 (purple trace)/TEN1 (orange trace)/CST (green trace) [lanes 6, 10, 14, and 18] as compared to Pol β synthesis alone (black trace) [lane 2].

Article Snippet: Primary antibodies: 1:100 mouse α-STN1 (Novus, NBP2-01006), 1:100 rabbit α-POLB (Abbexa, abx304879), 1:100 rabbit α-PolA1 (Bethyl Laboratories, A302-850A), 1:100 rabbit α-XRCC1 (Genetex, GTX111712), 1:100 mouse α-XRCC1 (Invitrogen, MA1-12640), 1:100 rabbit α-PARP1 (Proteintech, 6520-1-Ig), 1:100 rabbit α-OGG1 (Proteintech, 15125-1-AP).

Techniques: Generated, Concentration Assay

Journal: Journal of molecular biology

Article Title: Human CST Stimulates Base Excision Repair to Prevent the Accumulation of Oxidative DNA Damage

doi: 10.1016/j.jmb.2024.168672

Figure Lengend Snippet: (A) Substrate (5 nM) was pre-treated with 1 U of UDG before the addition of 100 nM APE1 to generate the dRP substrate, as shown in the schematic and described in “ ” (B) The lyase activity of Pol β (0.25 nM, lanes 1–13; 5 nM, lane 14) was observed in the presence of increasing concentrations of CTC1/STN1/TEN1 (250, 750, 1500 nM) or CST (7.5, 15, 45 nM) as denoted in the figure. Gel shown is representative of at least three replicate experiments. (C) Graph of the fold increase in lyase activity for the lanes containing the highest concentration of CST proteins utilized. The error bars represent the standard error mean. * denotes the dRP lyase substrate; ** denotes the dRP lyase product. The substrate was generated by annealing primers U7:T1 in a 1:2 ratio. (**** p < 0.0001).

Article Snippet: Primary antibodies: 1:100 mouse α-STN1 (Novus, NBP2-01006), 1:100 rabbit α-POLB (Abbexa, abx304879), 1:100 rabbit α-PolA1 (Bethyl Laboratories, A302-850A), 1:100 rabbit α-XRCC1 (Genetex, GTX111712), 1:100 mouse α-XRCC1 (Invitrogen, MA1-12640), 1:100 rabbit α-PARP1 (Proteintech, 6520-1-Ig), 1:100 rabbit α-OGG1 (Proteintech, 15125-1-AP).

Techniques: Activity Assay, Concentration Assay, Generated

Journal: Journal of molecular biology

Article Title: Human CST Stimulates Base Excision Repair to Prevent the Accumulation of Oxidative DNA Damage

doi: 10.1016/j.jmb.2024.168672

Figure Lengend Snippet: FEN1 (0.025 nM, lanes 2, 4–15; 10 nM, lane 2) cleavage activity was measured on a random sequence 5′ DNA flap DNA substrate (5 nM) in the presence of increasing concentrations of CTC1/STN1/TEN1 (250, 750, 1500 nM) or CST (5, 15, 60 nM) as denoted in the figure. The red asterisk shown on the substrate in the figure represents the location of the 32 P radiolabel. Gel shown is representative of at least three independent experiments. (B) Graph of the fold increase in FEN1 cleavage activity for each of the lanes containing the highest concentration of CST proteins utilized. (C) Shows the fold increase in FEN1 cleavage activity on a telomere substrate calculated as in (B). The error bars represent the standard error mean. The random substrate (U8:T5:D4) and telomere substrate (U9:T6:D5) was generated by annealing primers in a 4:2:1 ratio. (*** p < 0.001, ** p < 0.01, * p < 0.05).

Article Snippet: Primary antibodies: 1:100 mouse α-STN1 (Novus, NBP2-01006), 1:100 rabbit α-POLB (Abbexa, abx304879), 1:100 rabbit α-PolA1 (Bethyl Laboratories, A302-850A), 1:100 rabbit α-XRCC1 (Genetex, GTX111712), 1:100 mouse α-XRCC1 (Invitrogen, MA1-12640), 1:100 rabbit α-PARP1 (Proteintech, 6520-1-Ig), 1:100 rabbit α-OGG1 (Proteintech, 15125-1-AP).

Techniques: Activity Assay, Sequencing, Concentration Assay, Generated

Journal: Journal of molecular biology

Article Title: Human CST Stimulates Base Excision Repair to Prevent the Accumulation of Oxidative DNA Damage

doi: 10.1016/j.jmb.2024.168672

Figure Lengend Snippet: (A) LIGI (0.05 nM, lanes 2, 4–15; 100 nM, lane 3) ligation activity was measured on a nicked DNA substrate (5 nM) in the presence of increasing concentrations of CTC1/STN1/TEN1 (250, 750, 1500 nM) or CST (5, 15, 45 nM) as denoted. The red asterisk shown on the substrate in the figure represents the location of the 32 P radiolabel. Gel shown is representative of at least three independent experiments. (B) Graph of the fold increase in LIGI ligation activity for the lanes containing the highest concentration of CST proteins utilized. (C) Shows the fold increase in LIGI ligation activity on a telomere substrate calculated as in (B). The error bars represent the standard error mean. The random substrate (U10:T1:D6) and telomere substrate (U11:T4:D7) was generated by annealing primers in a 1:2:4 ratio. (** p < 0.01, * p < 0.05).

Article Snippet: Primary antibodies: 1:100 mouse α-STN1 (Novus, NBP2-01006), 1:100 rabbit α-POLB (Abbexa, abx304879), 1:100 rabbit α-PolA1 (Bethyl Laboratories, A302-850A), 1:100 rabbit α-XRCC1 (Genetex, GTX111712), 1:100 mouse α-XRCC1 (Invitrogen, MA1-12640), 1:100 rabbit α-PARP1 (Proteintech, 6520-1-Ig), 1:100 rabbit α-OGG1 (Proteintech, 15125-1-AP).

Techniques: Ligation, Activity Assay, Concentration Assay, Generated

Journal: Journal of molecular biology

Article Title: Human CST Stimulates Base Excision Repair to Prevent the Accumulation of Oxidative DNA Damage

doi: 10.1016/j.jmb.2024.168672

Figure Lengend Snippet: (A) The LP-BER pathway was reconstituted in vitro by combining APE1 (3 nM), Pol β (0.5 nM), FEN1 (0.01 nM), and LIGI (0.1 nM) on a random sequence abasic substrate containing a tetrahydrofuran (THF) residue (5 nM). An Φ represents a THF residue and a red asterisk represents the location of the 32 P radiolabel on the substrate. The ligation efficiency was monitored following the addition of CTC1/STN1/TEN1 (100, 500, 1000 nM) or CST complex (14.75, 29.5, 59 nM) as denoted. Gel shown is representative of at least three replicate experiments. (B) Graph of the fold increase in ligation efficiency for the lanes containing the highest concentration of CST proteins utilized. (C) Shows the fold increase in ligation efficiency on a telomere substrate calculated as in (B). The error bars represent the standard error mean. The random substrate (U2:T1) and telomere substrate (U3:T2) was generated by annealing primers in a 1:2 ratio. (**** p < 0.0001, ** p < 0.01).

Article Snippet: Primary antibodies: 1:100 mouse α-STN1 (Novus, NBP2-01006), 1:100 rabbit α-POLB (Abbexa, abx304879), 1:100 rabbit α-PolA1 (Bethyl Laboratories, A302-850A), 1:100 rabbit α-XRCC1 (Genetex, GTX111712), 1:100 mouse α-XRCC1 (Invitrogen, MA1-12640), 1:100 rabbit α-PARP1 (Proteintech, 6520-1-Ig), 1:100 rabbit α-OGG1 (Proteintech, 15125-1-AP).

Techniques: In Vitro, Sequencing, Residue, Ligation, Concentration Assay, Generated

Journal: Journal of molecular biology

Article Title: Human CST Stimulates Base Excision Repair to Prevent the Accumulation of Oxidative DNA Damage

doi: 10.1016/j.jmb.2024.168672

Figure Lengend Snippet: Parental and STN1 KO (A) or CTC1 KO (B) cells were treated with 0, 50, 100 or 200 μM H 2 O 2 for 1 h before release and recover for 4 h. Cells were collected, and genomic DNA extracted and 8-oxoG levels measured. A representative trial of at least two independent biological trials is shown. Circles represent three technical replicates. The error bars represent the standard error mean. (**** p < 0.0001, *** p < 0.001, ** p < 0.01).

Article Snippet: Primary antibodies: 1:100 mouse α-STN1 (Novus, NBP2-01006), 1:100 rabbit α-POLB (Abbexa, abx304879), 1:100 rabbit α-PolA1 (Bethyl Laboratories, A302-850A), 1:100 rabbit α-XRCC1 (Genetex, GTX111712), 1:100 mouse α-XRCC1 (Invitrogen, MA1-12640), 1:100 rabbit α-PARP1 (Proteintech, 6520-1-Ig), 1:100 rabbit α-OGG1 (Proteintech, 15125-1-AP).

Techniques:

Journal: Journal of molecular biology

Article Title: Human CST Stimulates Base Excision Repair to Prevent the Accumulation of Oxidative DNA Damage.

doi: 10.1016/j.jmb.2024.168672

Figure Lengend Snippet: Figure 1. CST associates with BER proteins. (A) Representative images of proximity ligation assay (PLA) performed in HeLa cells with antibodies to STN1 or Pol b alone or in combination with and without H2O2 treatment. Red, PLA foci; blue, DAPI. (B) Violin plot of PLA foci per nucleus. Results are representative of three independent, biological experiments. Bold dashed line represents the median, and dashed lines represent the first and third quartiles. (C) Proximity ligation assay (PLA) performed in HeLa cells with antibodies to STN1, OGG1, or in combination (STN1 + OGG1). n = 3 independent, biological experiments. Bold dashed line: median, dashed lines: first and third quartiles. (D) HEK293T cell extracts containing individually overexpressed CST components (either FLAG- CST, Myc-STN1 or FLAG-TEN1) that were either untreated or treated with 200 mM of H2O2 were used to immunoprecipitate APE1, Pol b, FEN1 or LIGI and immunobloted with either anti-FLAG (for CTC1 and TEN1) or anti- Myc for STN1. Lysates were pre-treated with benzonase prior to coimmunoprecipitation. Proteins are identified in the Western blot analysis. (****p < 0.0001).

Article Snippet: Primary antibodies: 1:100 mouse a-STN1 (Novus, NBP2-01006), 1:100 rabbit a-POLB (Abbexa, abx304879), 1:100 rabbit a-PolA1 (Bethyl Laboratories, A302-850A), 1:100 rabbit a-XRCC1 (Genetex, GTX111712), 1:100 mouse a-XRCC1 (Invitrogen, MA1-12640), 1:100 rabbit a-PARP1 (Proteintech, 6520-1-Ig), 1:100 rabbit a-OGG1 (Proteintech, 15125-1-AP).

Techniques: Proximity Ligation Assay, Western Blot

Journal: Journal of molecular biology

Article Title: Human CST Stimulates Base Excision Repair to Prevent the Accumulation of Oxidative DNA Damage.

doi: 10.1016/j.jmb.2024.168672

Figure Lengend Snippet: Figure 2. The CST complex and individual subunits stimulate OGG1 activity on 8-oxo-G containing substrate. (A) OGG1 (2 nM, lanes 2, 4–15; 50 nM, lane 3) lesion base removal and phosphodiester bone cleavage activity were measured on a random sequence DNA substrate (5 nM) containing an 8-oxo-guanine (8-oxo-G) residue in the presence of increasing concentrations of CTC1/STN1/TEN1 (250, 750, 1500 nM) or CST (5, 15, 60 nM) as denoted in the figure. The red asterisk shown on the substrate in the figure represents the location of the 32P radiolabel and a G* represents an 8-oxo-G residue. Gel shown is representative of at least three independent experiments. (B) Graph of the fold increase in OGG1 cleavage activity for the lanes containing the highest concentration of the CST complex and individual subunits. The random substrate (U1:T1) was generated by annealing primers in a 1:2 ratio. (****p < 0.0001).

Article Snippet: Primary antibodies: 1:100 mouse a-STN1 (Novus, NBP2-01006), 1:100 rabbit a-POLB (Abbexa, abx304879), 1:100 rabbit a-PolA1 (Bethyl Laboratories, A302-850A), 1:100 rabbit a-XRCC1 (Genetex, GTX111712), 1:100 mouse a-XRCC1 (Invitrogen, MA1-12640), 1:100 rabbit a-PARP1 (Proteintech, 6520-1-Ig), 1:100 rabbit a-OGG1 (Proteintech, 15125-1-AP).

Techniques: Activity Assay, Sequencing, Residue, Concentration Assay, Generated

Journal: Journal of molecular biology

Article Title: Human CST Stimulates Base Excision Repair to Prevent the Accumulation of Oxidative DNA Damage.

doi: 10.1016/j.jmb.2024.168672

Figure Lengend Snippet: Figure 3. The CST complex and CTC1 enhance APE1 cleavage activity. (A) APE1 (0.075 nM, lanes 2, 4–15; 100 nM, lane 3) cleavage activity was measured on a random sequence DNA substrate (5 nM) containing a THF residue in the presence of increasing concentrations of CTC1/STN1/TEN1 (250, 750, 1500 nM) or CST (5, 15, 60 nM) as denoted in the figure. The red asterisk shown on the substrate in the figure represents the location of the 32P radiolabel and an U represents a THF residue. Gel shown is representative of at least three independent experiments. (B) Graph of the fold increase in APE1 cleavage activity for the lanes containing the highest concentration of the CST complex and individual subunits. (C) Graph of the fold increase in APE1 cleavage activity on a telomere substrate calculated as in (B). The error bars represent the standard error mean. The random substrate (U2:T1) and telomere substrate (U3:T2) was generated by annealing primers in a 1:2 ratio. (****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05).

Article Snippet: Primary antibodies: 1:100 mouse a-STN1 (Novus, NBP2-01006), 1:100 rabbit a-POLB (Abbexa, abx304879), 1:100 rabbit a-PolA1 (Bethyl Laboratories, A302-850A), 1:100 rabbit a-XRCC1 (Genetex, GTX111712), 1:100 mouse a-XRCC1 (Invitrogen, MA1-12640), 1:100 rabbit a-PARP1 (Proteintech, 6520-1-Ig), 1:100 rabbit a-OGG1 (Proteintech, 15125-1-AP).

Techniques: Activity Assay, Sequencing, Residue, Concentration Assay, Generated

Journal: Journal of molecular biology

Article Title: Human CST Stimulates Base Excision Repair to Prevent the Accumulation of Oxidative DNA Damage.

doi: 10.1016/j.jmb.2024.168672

Figure Lengend Snippet: Figure 4. The CST complex and individual subunits stimulate DNA Pol b synthesis on a gap substrate containing an abasic site. Synthesis by DNA Pol b (2 nM) on a 1 nt gapped substrate (5 nM) containing an abasic site mimic (U denotes tetrahydrofuran [THF]) in the presence of increasing amounts of CTC1/STN1/TEN1 (100, 500, 1000 nM) or CST (5, 15, 30 nM) as denoted in the figure. The substrate was generated by annealing primers U4:T1: D1 in a 1:2:4 ratio. The red asterisk shown on the substrate represents the location of the 32P radiolabel. Gel shown is representative of at least three independent experiments. A darker exposure of the gel shows the strand displacement synthesis products. (B) The trace at the right of the gel depicts the signal intensity of the gap-filling and strand- displacement products in the lanes containing the highest concentration of CTC1(red trace)/STN1 (purple trace)/ TEN1 (orange trace)/CST (green trace) [lanes 6, 10, 14, and 18] as compared to Pol b synthesis alone (black trace) [lane 2].

Article Snippet: Primary antibodies: 1:100 mouse a-STN1 (Novus, NBP2-01006), 1:100 rabbit a-POLB (Abbexa, abx304879), 1:100 rabbit a-PolA1 (Bethyl Laboratories, A302-850A), 1:100 rabbit a-XRCC1 (Genetex, GTX111712), 1:100 mouse a-XRCC1 (Invitrogen, MA1-12640), 1:100 rabbit a-PARP1 (Proteintech, 6520-1-Ig), 1:100 rabbit a-OGG1 (Proteintech, 15125-1-AP).

Techniques: Generated, Concentration Assay

Journal: Journal of molecular biology

Article Title: Human CST Stimulates Base Excision Repair to Prevent the Accumulation of Oxidative DNA Damage.

doi: 10.1016/j.jmb.2024.168672

Figure Lengend Snippet: Figure 5. The CST complex, CTC1, and STN1 subunits stimulate dRP lyase activity of Pol b. (A) Substrate (5 nM) was pre-treated with 1 U of UDG before the addition of 100 nM APE1 to generate the dRP substrate, as shown in the schematic and described in “Materials and Methods” (B) The lyase activity of Pol b (0.25 nM, lanes 1–13; 5 nM, lane 14) was observed in the presence of increasing concentrations of CTC1/STN1/TEN1 (250, 750, 1500 nM) or CST (7.5, 15, 45 nM) as denoted in the figure. Gel shown is representative of at least three replicate experiments. (C) Graph of the fold increase in lyase activity for the lanes containing the highest concentration of CST proteins utilized. The error bars represent the standard error mean. * denotes the dRP lyase substrate; ** denotes the dRP lyase product. The substrate was generated by annealing primers U7:T1 in a 1:2 ratio. (****p < 0.0001).

Article Snippet: Primary antibodies: 1:100 mouse a-STN1 (Novus, NBP2-01006), 1:100 rabbit a-POLB (Abbexa, abx304879), 1:100 rabbit a-PolA1 (Bethyl Laboratories, A302-850A), 1:100 rabbit a-XRCC1 (Genetex, GTX111712), 1:100 mouse a-XRCC1 (Invitrogen, MA1-12640), 1:100 rabbit a-PARP1 (Proteintech, 6520-1-Ig), 1:100 rabbit a-OGG1 (Proteintech, 15125-1-AP).

Techniques: Activity Assay, Concentration Assay, Generated

Journal: Journal of molecular biology

Article Title: Human CST Stimulates Base Excision Repair to Prevent the Accumulation of Oxidative DNA Damage.

doi: 10.1016/j.jmb.2024.168672

Figure Lengend Snippet: Figure 6. FEN1 cleavage activity is stimulated by CTC1 but not the full CST complex. FEN1 (0.025 nM, lanes 2, 4–15; 10 nM, lane 2) cleavage activity was measured on a random sequence 50 DNA flap DNA substrate (5 nM) in the presence of increasing concentrations of CTC1/STN1/TEN1 (250, 750, 1500 nM) or CST (5, 15, 60 nM) as denoted in the figure. The red asterisk shown on the substrate in the figure represents the location of the 32P radiolabel. Gel shown is representative of at least three independent experiments. (B) Graph of the fold increase in FEN1 cleavage activity for each of the lanes containing the highest concentration of CST proteins utilized. (C) Shows the fold increase in FEN1 cleavage activity on a telomere substrate calculated as in (B). The error bars represent the standard error mean. The random substrate (U8:T5:D4) and telomere substrate (U9:T6:D5) was generated by annealing primers in a 4:2:1 ratio. (***p < 0.001, **p < 0.01, *p < 0.05).

Article Snippet: Primary antibodies: 1:100 mouse a-STN1 (Novus, NBP2-01006), 1:100 rabbit a-POLB (Abbexa, abx304879), 1:100 rabbit a-PolA1 (Bethyl Laboratories, A302-850A), 1:100 rabbit a-XRCC1 (Genetex, GTX111712), 1:100 mouse a-XRCC1 (Invitrogen, MA1-12640), 1:100 rabbit a-PARP1 (Proteintech, 6520-1-Ig), 1:100 rabbit a-OGG1 (Proteintech, 15125-1-AP).

Techniques: Activity Assay, Sequencing, Concentration Assay, Generated

Journal: Journal of molecular biology

Article Title: Human CST Stimulates Base Excision Repair to Prevent the Accumulation of Oxidative DNA Damage.

doi: 10.1016/j.jmb.2024.168672

Figure Lengend Snippet: Figure 7. The CST complex, CTC1 and TEN1 stimulate ligation efficiency of LIGI. (A) LIGI (0.05 nM, lanes 2, 4–15; 100 nM, lane 3) ligation activity was measured on a nicked DNA substrate (5 nM) in the presence of increasing concentrations of CTC1/STN1/TEN1 (250, 750, 1500 nM) or CST (5, 15, 45 nM) as denoted. The red asterisk shown on the substrate in the figure represents the location of the 32P radiolabel. Gel shown is representative of at least three independent experiments. (B) Graph of the fold increase in LIGI ligation activity for the lanes containing the highest concentration of CST proteins utilized. (C) Shows the fold increase in LIGI ligation activity on a telomere substrate calculated as in (B). The error bars represent the standard error mean. The random substrate (U10:T1:D6) and telomere substrate (U11:T4:D7) was generated by annealing primers in a 1:2:4 ratio. (**p < 0.01, *p < 0.05).

Article Snippet: Primary antibodies: 1:100 mouse a-STN1 (Novus, NBP2-01006), 1:100 rabbit a-POLB (Abbexa, abx304879), 1:100 rabbit a-PolA1 (Bethyl Laboratories, A302-850A), 1:100 rabbit a-XRCC1 (Genetex, GTX111712), 1:100 mouse a-XRCC1 (Invitrogen, MA1-12640), 1:100 rabbit a-PARP1 (Proteintech, 6520-1-Ig), 1:100 rabbit a-OGG1 (Proteintech, 15125-1-AP).

Techniques: Ligation, Activity Assay, Concentration Assay, Generated

Journal: Journal of molecular biology

Article Title: Human CST Stimulates Base Excision Repair to Prevent the Accumulation of Oxidative DNA Damage.

doi: 10.1016/j.jmb.2024.168672

Figure Lengend Snippet: Figure 8. The CST complex, CTC1 and STN1 increase the efficiency of LP-BER. (A) The LP-BER pathway was reconstituted in vitro by combining APE1 (3 nM), Pol b (0.5 nM), FEN1 (0.01 nM), and LIGI (0.1 nM) on a random sequence abasic substrate containing a tetrahydrofuran (THF) residue (5 nM). An U represents a THF residue and a red asterisk represents the location of the 32P radiolabel on the substrate. The ligation efficiency was monitored following the addition of CTC1/STN1/TEN1 (100, 500, 1000 nM) or CST complex (14.75, 29.5, 59 nM) as denoted. Gel shown is representative of at least three replicate experiments. (B) Graph of the fold increase in ligation efficiency for the lanes containing the highest concentration of CST proteins utilized. (C) Shows the fold increase in ligation efficiency on a telomere substrate calculated as in (B). The error bars represent the standard error mean. The random substrate (U2:T1) and telomere substrate (U3:T2) was generated by annealing primers in a 1:2 ratio. (****p < 0.0001, **p < 0.01).

Article Snippet: Primary antibodies: 1:100 mouse a-STN1 (Novus, NBP2-01006), 1:100 rabbit a-POLB (Abbexa, abx304879), 1:100 rabbit a-PolA1 (Bethyl Laboratories, A302-850A), 1:100 rabbit a-XRCC1 (Genetex, GTX111712), 1:100 mouse a-XRCC1 (Invitrogen, MA1-12640), 1:100 rabbit a-PARP1 (Proteintech, 6520-1-Ig), 1:100 rabbit a-OGG1 (Proteintech, 15125-1-AP).

Techniques: In Vitro, Sequencing, Residue, Ligation, Concentration Assay, Generated

Journal: Journal of molecular biology

Article Title: Human CST Stimulates Base Excision Repair to Prevent the Accumulation of Oxidative DNA Damage.

doi: 10.1016/j.jmb.2024.168672

Figure Lengend Snippet: Figure 9. CST prevents the accumulation of genomic 8-oxo-Gs. Parental and STN1 KO (A) or CTC1 KO (B) cells were treated with 0, 50, 100 or 200 lM H2O2 for 1 h before release and recover for 4 h. Cells were collected, and genomic DNA extracted and 8-oxoG levels measured. A representative trial of at least two independent biological trials is shown. Circles represent three technical replicates. The error bars represent the standard error mean. (****p < 0.0001, ***p < 0.001, **p < 0.01).

Article Snippet: Primary antibodies: 1:100 mouse a-STN1 (Novus, NBP2-01006), 1:100 rabbit a-POLB (Abbexa, abx304879), 1:100 rabbit a-PolA1 (Bethyl Laboratories, A302-850A), 1:100 rabbit a-XRCC1 (Genetex, GTX111712), 1:100 mouse a-XRCC1 (Invitrogen, MA1-12640), 1:100 rabbit a-PARP1 (Proteintech, 6520-1-Ig), 1:100 rabbit a-OGG1 (Proteintech, 15125-1-AP).

Techniques: