![L557P causes a defect in telomerase assembly, whereas K1050E affects binding of telomerase to its DNA substrate. (A) Northern dot-blot showing the amount of hTR recovered after IP of hTERT from lysates of HEK293T cells expressing the indicated hTERT variants. Samples were normalized to the recovery of hTERT on western blots and loaded in duplicate, with their average used for quantitation. (B) Quantitation of the amount of hTERT-hTR binding from panel A; data are the mean ± standard error of the mean (SEM; n = 4). *P = .0193, by repeated measures 1-way analysis of variance (ANOVA), followed by Dunnett’s post hoc test. (C) DNA binding assay to determine primer affinity (KD) of telomerase variants. Purified human telomerase was incubated with a biotinylated DNA oligonucleotide primer (biotin-TTAGGG)3) at the indicated primer concentrations, and the amount of telomerase remaining in the supernatant after recovery of the DNA on NeutrAvidin beads was quantitated by northern dot-blot for hTR; more telomerase was bound at higher DNA concentrations and removed from the solution, leaving less free telomerase in the supernatant. (D) Quantitation of telomerase primer KD for the indicated hTERT variants. Plots of the amount of telomerase removed from the solution were fitted to the equation y = (Bmax[S])/(KD + [S]), where Bmax is the maximum level of binding, [S] is the concentration of DNA, and KD is the equilibrium binding constant. Data are the mean ± SEM (n = 3-7); ***P = .001, by ordinary 1-way ANOVA, followed by Dunnett’s post hoc test.](https://ash.silverchair-cdn.com/ash/content_public/journal/bloodadvances/6/12/10.1182_bloodadvances.2022007029/1/advancesadv2022007029f4.png?Expires=1724963151&Signature=TKdFbWutP6L8PLy~6vE4U84RBptDHprVrfnUynr64rKn~Ui7Ih52vc36AEzv8PzRYb-SvpyFjfg17fa-5R9hbQxVeHZpE1T8-MZrIqQgCKltBs7pWjRL~7MEKP-S8hFL5P8Z6dWUbGwd4X7ATP7AoVGy9aYyvqYDpT3Sc~CkusL8mDTLtYi1QbXjzkV-aMc1Ma~X80Q0-8qLbufyLstGnOAQsz-m9L8BqCrwFdE7ECpK4LK7LUP9g0KHiYAJSCn~GcJoXJveeKfP31x52GjkONDdNL~kXeim8uc4c-mxInqmohF8Df7gNu6E~PLW2R2EPK0Tj1s94aXYJDvgXTyrAA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
L557P causes a defect in telomerase assembly, whereas K1050E affects binding of telomerase to its DNA substrate. (A) Northern dot-blot showing the amount of hTR recovered after IP of hTERT from lysates of HEK293T cells expressing the indicated hTERT variants. Samples were normalized to the recovery of hTERT on western blots and loaded in duplicate, with their average used for quantitation. (B) Quantitation of the amount of hTERT-hTR binding from panel A; data are the mean ± standard error of the mean (SEM; n = 4). *P = .0193, by repeated measures 1-way analysis of variance (ANOVA), followed by Dunnett’s post hoc test. (C) DNA binding assay to determine primer affinity (KD) of telomerase variants. Purified human telomerase was incubated with a biotinylated DNA oligonucleotide primer (biotin-TTAGGG)3) at the indicated primer concentrations, and the amount of telomerase remaining in the supernatant after recovery of the DNA on NeutrAvidin beads was quantitated by northern dot-blot for hTR; more telomerase was bound at higher DNA concentrations and removed from the solution, leaving less free telomerase in the supernatant. (D) Quantitation of telomerase primer KD for the indicated hTERT variants. Plots of the amount of telomerase removed from the solution were fitted to the equation y = (Bmax[S])/(KD + [S]), where Bmax is the maximum level of binding, [S] is the concentration of DNA, and KD is the equilibrium binding constant. Data are the mean ± SEM (n = 3-7); ***P = .001, by ordinary 1-way ANOVA, followed by Dunnett’s post hoc test.