Fig. 3: EP300 deficiency results in genome-wide replication stalling and fork collapse, leading to the accumulation of extensive ssDNA gaps. | Nature Communications

Fig. 3: EP300 deficiency results in genome-wide replication stalling and fork collapse, leading to the accumulation of extensive ssDNA gaps.

From: EP300 deficiency leads to chronic replication stress mediated by defective replication fork protection

Fig. 3

A Schematic model depicting replication fork stalling, fork protection, and nucleolytic degradation, (Created in BioRender. Madireddy, A. (2025), is licensed under CC BY 4.0, https://BioRender.com/xtm4wme). B DNA fiber analysis of 50uM hydroxyurea (HU) (60 min) treated EP300WT (NA1, NA4), EP300Mut (NA2, NA3) and EP300WT NA-ATLL (NA1, NA4) cells treated with negative control (S, S) stereoisomer or with JQAD1 (500 nM for 48 h) treatment to assess replication fork stalling. The fork rate (CldU/IdU ratio) is indicated. N = 3. C DNA fiber analysis measuring nucleolytic degradation after 4 mM HU treatment in EP300WT, EP300Mut NA-ATLL cells. The fork rate (CldU/IdU ratio) is indicated, N = 3. D Analysis of the number of single-stranded gaps (ssDNA)/Iododeoxyuridine (IdU) foci (green) per cell nuclei (DAPI, blue) in EP300WT (NA4) and EP300Mut (NA2, NA3) cells exposed to 2 mM HU (4 h), and EP300WT (NA4) cells treated with JQAD1 (500 nM). ssDNA gaps are measured by IdU incorporation under non-denaturing conditions. Representative images are shown on the right, N = 3. E Analysis of number of pRPA Ser4/8 foci (red) per cell nuclei (DAPI, blue) in EP300WT (NA4) and EP300Mut (NA2, NA3) cells exposed to 2 mM HU (4 h), and EP300WT (NA4) cells treated with 500 nM JQAD1. Representative images are shown on the right, N = 3. F Protein expression of phospho-RPA Ser4/8 in the presence or absence of EP300, presented as a fold change quantified from the immunoblot in Supp. Fig. S3D. G Analysis of the number of single-stranded gaps/breaks (ssDNA)/Iododeoxyuridine (IdU) foci per cell nucleus (DAPI, blue) in EP300WT (NA4) and EP300Mut (NA3) cells exposed to 2 mM HU (4 h), in the presence or absence of 50uM Mirin (4 h) (Mre11 nuclease inhibitor), N = 3. H Analysis of the number of single-stranded gaps/breaks (ssDNA)/ Iododeoxyuridine (IdU) foci per cell nucleus (DAPI, blue) in EP300WT (NA4) and EP300Mut (NA3) cells exposed to 2 mM HU (4 h), in the presence or absence of 20uM C5 (24 h) (DNA2 nuclease inhibitor), N = 3. For all experiments, data are presented as mean values +/− SD. P-values were determined by a two-tailed Student T-test. P-values are indicated as follows: * <0.03, ** <0.0021, *** <0.0002, **** <0.0001. Scale bar 10 μm. N represents three experimental replicates from independent cultures of cells. Source data are provided as a Source Data file.

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