Fig. 5
From: Telomerase subunit Est2 marks internal sites that are prone to accumulate DNA damage
Est2 binding is affected by DNA damage. A ChIP-qPCR of γ-H2A-binding to NTBS regions demonstrating their DNA damage prone nature. H2A-binding to four NTBS (#1-#4) and compared to S129A mutant (no γ-H2A phosphorylation). Data plotted are mean ± SEM for n = 3 biological replicates with wild type (light grey bars) and S129 mutant (dark grey bars) conditions. Statistical significance compared to S129 mutant conditions were determined using Student’s t-test. **p-value < 0.01. B Telomere addition frequency was determined in undamaged (light grey bars) and in damage (IR, dark grey bars) and was calculated as described before [26]. For IR treatment, cells were irradiated at 20 Gy before crosslinking and immunoprecipitated using the standard procedures mentioned in the methods. Telomere addition frequency was measured using a genetic assay based on loss of distal LYS2 gene (resistance to α-aminoadipate). TG80 and N80 were used as positive and negative control. TG80 contains 80 bp TG1–3 ; N80 contains 80 bp lambda DNA. C–E ChIP analysis of Est2, Est1, and Est3 to four NTBS and one telomere (VI-R) in undamaged (light grey bars) and damaging (IR, dark grey bars) conditions). For IR treatment, cells were irradiated at 20 Gy before crosslinking and immunoprecipitated using the standard procedures mentioned in the methods. C Est2-binding to NTBS, ARO1 and non-γ-H2A regions. Data plotted are IP/Input values represented as mean ± SEM of n = 3 biological replicates. Statistical significance compared to untreated cells were determined using Student’s t-test. *p-value < 0.05 and **p-value < 0.01. D Est1-NTBS-binding. ChIP is normalized to ARO1 and represented as mean ± SEM. E Est3-NTBS-binding in undamaged (light grey bars) and damaging (IR, dark grey bars) conditions. Bars represent enrichment over ARO1. Data are represented as mean ± SEM for n = 3 biological replicates. Statistical significance compared to untreated cells were determined using Student’s t-test. *p-value < 0.05 and **p-value < 0.01. F Quantification of Est2-binding upon induction of cleavage at the HO site. Est2-binding by ChIP to NTBS near HO cut sites was monitored before (light grey bars) and after induction (dark grey bars) of HO endonuclease. Data were plotted as mean ± SEM of n = 3 biological replicates. Statistical tests were performed by comparing induced to uninduced conditions and were determined using Student’s t-test. ** p-value < 0.01