Fig. 2: 5hmdC-induced DNA damage associates to S-phase and impairs replication fork progression. | Cell Death & Disease

Fig. 2: 5hmdC-induced DNA damage associates to S-phase and impairs replication fork progression.

From: FANCD2 maintains replication fork stability during misincorporation of the DNA demethylation products 5-hydroxymethyl-2’-deoxycytidine and 5-hydroxymethyl-2’-deoxyuridine

Fig. 2

A HPLC-MS/MS quantitation of endogenous and exogenous 5hmdC levels in genomic DNA samples of wild type and Fancd2−/− cells exposed to D3-labeled 5hmdC (0, 10 and 20 μM) for 16 h. Wild type cells showed a 0.49 vs 4.8 exogenous vs endogenous 5hmdC per 104 dG upon 10 μM 5hmdC-D3 exposure; 1.2 5hmdC-D3 vs 4.5 5hmdC per 104 dG for 20 μM exposure). Fancd2−/− cells showed a 3.2 5hmdC-D3 vs 5.8 5hmdC per 104 dG upon 10 μM 5hmdC-D3 exposure; 2.82 5hmdC-D3 vs 4.91 5hmdC per 104 dG upon 20 μM 5hmdC-D3 exposure. Plots represent mean values from 3 biological replicates. B Left, representative immunofluorescence of wild type and Fancd2−/− MEFs showing PAR nuclear staining and EdU positive (EdU+) cells after exposure to 5hmdC (10 μM) for 16 h (n = 3). Right, bar plot showing percentage of PAR+ cells in EdU negative (EdU−) or EdU+ cell populations (n = 250 of each of 3 independent biological replicates, Student’s t test; bars represents mean ± s.d.). C Western blot of iPOND-captured and input protein extracts from wild type and Fancd2−/− cells treated with 5hmdC (160 μM) or HU (1 mM) for 3 h to detect γ-H2AX, PCNA and RAD51. D Top left, scheme of BrdU incorporation assay. Bottom left, representative immunofluorescence images of wild type and Fancd2−/− MEFs showing BrdU + (Red) or BrdU- cells and nuclear DNA (DAPI, blue). Right, percentage of BrdU+ or BrdU− cells after HU (1 mM) or 5hmdC (40 μM) for 16 h (n = 200 of each of 3 biological replicates, Student’s t test; Bar represents mean ± s.d.). E Top, plot depicting PAR mean intensity signal per nucleus of wild type and Fancd2−/− cells exposed to 10, 40, 80 or 160 μM 5hmdC for 1 h (n = 3, Mann–Whitney test; central line represents median value). Bottom, plot depicting γ-H2AX foci per nucleus of wild type and Fancd2−/− cells exposed to 10, 40, 80 or 160 μM 5hmdC for 1 h (n = 3, Student’s t test; central line represents mean ± s.d.). F Top left, scheme of the DNA fiber assay. Bottom left, representative images of DNA fibers from wild type and Fancd2−/− MEFs after 10, 40, 80 or 160 μM 5hmdC exposure. Right, plot representing fork speed (Kbp min−1) of wild type and Fancd2−/− MEFs exposed to 10, 40, 80 or 160 μM 5hmdC (n = 150 of each of 3 biological replicates, Mann–Whitney test; central line represents median value).

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