Fig 1: Microhomology-mediated strand transfer and DNA synthesis by DNA polymerase ? on telomere sequences is regulated by RP-A and POT1/TPP1. DNA templates are shown on top of each panel. Black boxes indicate the annealing site on the 5'-labelled primer and template strands. (A) Titration of Pol ? alone (lanes 1–5; 11–15) or in combination with 25 nM RP-A (lanes 6–10; 16–20), in the presence of the 16/48 linear (lanes 1–10) or the 48/48 “bubble” (lanes 11–20) acceptor DNA templates. (B) Titration of dideoxy-terminated 16/48mer (lanes 2–11) or 48/48mer “bubble” (lanes 12–21) acceptor DNA templates, in the presence of dGTP and in the absence (lanes 2–6; 12–16) or in the presence (lanes 7–11; 17–21) of 25 nM RP-A. (C) Dose-response curves of nucleotide incorporation (pmols) by Pol ? in the absence (clear symbols) or presence (filled symbols) of 25 nM RP-A on the linear (circles) or bubble (triangles) substrates illustrated in panel B. Values are the mean of two measurements ± S.E. (D) Titration of Pol ? on the 48/48mer “bubble” template, in the absence (lanes 1–4) or in the presence (lanes 5–12) of different amounts of the POT1/TPP1 complex. (E) Titration of Pol ? on the 48/48mer “bubble” template (25 nM), in the absence (lanes 11–13) or in the presence (lanes 1–10) of different amounts of the POT1/TPP1 complex. Lane 1: control in the absence of enzyme. (F) Quantification of nucleotide incorporation by increasing amounts of Pol ? in the absence (filled circles) or in the presence of increasing amounts of POT1/PP1 as indicated in panel E. Values are the mean of two measurements ±S.E. (G) Pol ? activity on the 48/48 mer acceptor template in the absence (lane 2) or in the presence (lanes 3–6) of increasing amounts of POT1/TPP1. (H) Titration of the 48/48 mer acceptor template in the presence of Pol ? and in the presence (lanes 1–5) or in the absence (lanes 6–9) of POT1/TPP1. (I) Dose-response curves of nucleotide incorporation (pmols) by Pol ? in the absence (clear symbols) or presence (filled symbols) of 15 nM POT1/TTP1 on the bubble substrate shown in panel H. Values are the mean of two measurements ± S.E.
Fig 2: TERRA RNA represses the strand transfer activity by DNA polymerase ? and is counteracted by POT1/TPP1. (A) Time course of MMST synthesis by Pol ? with the 26 mer donor DNA (lanes 1–6) or the 26 mer TERRA donor RNA (lanes 8–14) and the 5xtel 16/48 mer acceptor template. Lane 7, reaction with the 26 mer donor DNA without the acceptor template. Lane 15, reaction with the 26 mer TERRA donor RNA without the acceptor template. (B) Titration of 26mer TERRA (lanes 2–8) or the 26mer DNA (lanes 9–16) in the DNA-dependent (lanes 2–8) or TERRA-dependent (lanes 9–16) MMST reaction catalyzed by Pol ? on the 48/48mer bubble acceptor template. (C) Dose response curves of the inhibition of Pol ? synthesis by the competitor 26mer TERRA (circles) or 26mer DNA (triangles) shown in panel B. Values are the mean of two measurements ± S.E. (D) Titration of 26mer TERRA in the DNA-dependent MMST reaction catalyzed by Pol ? on the 48/48mer bubble acceptor template, in the absence (lanes 2–6) or presence (lanes 7–21) of increasing POT1/TPP1 concentrations. (E) Dose response curves of the inhibition of Pol ? synthesis by the competitor 26mer TERRA, in the absence (triangles) or in the presence of increasing amounts of POT1/TPP1. Values are the mean of two measurements ± S.E.
Fig 3: DNA polymerase ? silencing causes telomere stress specifically in ALT cells. (A) Confocal microscopy imaging of TPP1 colocalization with P53BP1 (telomere dysfunction foci, TIF) in Saos-2 cells silenced for Pol ?. The white arrows indicate representative colocalization events (TIF). (B) Quantification of TPP1-P53BP1 colocalization events through JACoP plug-in of ImageJ. 103 and 106 cells were analyzed, respectively, for cells treated with scramble control siRNA (Control) and for cells silenced for Pol ? (siPolL). Each circle and triangle represents the amount of colocalizing signal within a single cell. These data are quantified by M2 Mander’s overlap coefficients, that are 0.7702 (SD = 0.06734) for cells treated with scrambled control siRNA and 0.8537 (SD = 0.06885) for cells silenced for Pol ?. Error bars represent ±SD. p-values were calculated through two-tailed Student’s t-test (95% confidence interval) (C) Confocal microscopy imaging of TPP1 colocalization with P53BP1 (telomere dysfunction foci, TIF) in U2OS cells silenced for Pol ?. The white arrows indicate representative colocalization events (TIF). (D) Statistical analysis comparing the number of TPP1-P53BP1 colocalization events in U2OS treated with scrambled control siRNA (control) or treated with anti-Pol ? siRNA (siPolL). n. of analyzed cells = 100 for each condition. ?2 p value is shown on top of the bars. (E) Statistical analysis comparing the number of TPP1-P53BP1 colocalization events in BJ-hTERT cells treated with scrambled control siRNA (control) or treated with anti-Pol ? siRNA (siPolL). n. of analyzed cells = 53 for each condition. ?2 p value is shown on top of the bars. n.s., not significant, ?2 p > 0.02. ** p value = 0.002, **** p value < 0.0001.
Fig 4: DNA polymerase ? associates with TPP1 in ALT cells (A-D Saos-2; E-H U2OS). (A) Confocal microscopy imaging of Pol ? colocalization with TPP1 in Saos-2 cells stably expressing c-Myc Pol ?. The white arrow indicates a representative colocalization event. (B) Number of foci detected for Myc-Pol ? (circles) and TPP1 (tringles) per cell. (C) Number of observed colocalization events per cell. (D) Statistical analysis comparing the expected number (n) of Pol ?-TPP1 colocalization events under the hypothesis of random chance colocalization, with the number of observed colocalizations. ?2 p values are shown on top of the bars. (E) Confocal microscopy imaging of Pol ? colocalization with TPP1 in U2OS cells. The white arrow indicates representative colocalization events in a single stack. (F) Number of observed foci for Pol ? (circles) and TPP1 (triangles) per cell. (G) Number of observed colocalization events per cell. (H) Statistical analysis comparing the expected number (n) of Pol ?-TPP1 colocalization events under the hypothesis of random chance colocalization, with the number of observed colocalizations. ?2 p values are shown on top of the bars. ***, ?2 p value < 0.005.
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