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. Author manuscript; available in PMC: 2016 Oct 25.
Published in final edited form as: Nat Cell Biol. 2016 Apr 25;18(6):684–691. doi: 10.1038/ncb3344

Figure 4. Overwound centromeric DNA suppresses RPA accumulation and ATR activation.

Figure 4

(a) EM of B18 DNA isolated after 150 minutes incubation in interphase extract. Arrows indicate ssDNA bubbles. Scheme is included. Data represent 1 out of 3 experiments. (b) Streptavidin-HRP stained dot blot of L10 ad B18 DNA isolated from BAC nuclei replicated with 40 μM topotecan (TPT) or left untreated and treated with biotin-psoralen (bPsoralen) in the presence (+) or absence (-) of UV. Data represent 1 out of 3 experiments (c) Total incorporated bPsoralen was measured using a fluorescence based biotin quantification assay. Values represent fluorescence intensity expressed in arbitrary units. Error bars represent ± sd of the mean. n=3 experiments; p<0.05 when comparing L10 and B18 mean values for all treatments; One-way Anova. (d) WB of RPA32 and MCM7 bound to B18 chromatin isolated from nuclei replicated in the presence of 40 μM topotecan (TPT) and 25 μg ml-1 APH as indicated. (e) RPA32/MCM7 ratio. Error bars represent ± sd of the mean. n=3 experiments; p<0.01 when comparing B18 mean values for all treatments; One-way Anova. (f) Average incorporation of 32P in histone H2AX C-terminal peptide in the presence of nucleoplasm derived from L10 and B18 nuclei replicated in extracts that were untreated (UN), treated with 25 μg ml-1 APH (High APH), 40 μM TPT, TPT and 1 μg ml-1 APH (TPT + Low APH) or TPT and 25 μg ml-1 APH (TPT + High APH). Reactions were performed in the absence (black bars) or in the presence (white bars) of 10 μM VE-821 (ATRi). Error bars represent ± sd of the mean. n=3 experiments; p<0.001 when comparing B18 and L10 mean values for all treatments; One-way Anova. (g) L10 and B18 DNA replication in extracts treated with 40 μM TPT and 1 μg ml-1 APH (Low APH) in the presence or absence of ATRi as indicated. Relative average percentage of 32PdCTP incorporation is shown. L10 values in untreated extracts were considered 100%. Error bars represent ± sd of the mean. n=3 experiments; p<0.001 when comparing B18 and L10 mean values for all treatments; One-way Anova.