Figure 6. RECQ1 associates with damaged chromatin in PARP activity-independent manner.

Sequential extraction of HeLa cells without and with H₂O₂ treatment. CSK and EXT fractions contain cytoplasmic, cytoskeletal, and soluble nuclear proteins. Insoluble nuclear pellet fractions contain proteins that are tightly bound to chromatin and/or the nuclear matrix. GAPDH and histone H3 serve as loading controls and markers for the fractions containing soluble proteins and chromatin, respectively. Results were documented using different exposures of the Western blots. A. RECQ1 associates with chromatin in a H₂O₂ dose-dependent manner. HeLa cells were treated with the indicated concentrations of H₂O₂ for 20 min at 37°C followed by sequential fractionation. B. H₂O₂ dose-dependent chromatin association of RECQ1 is accompanied by enrichment of PARP-1 on damaged chromatin. Nuclear pellet fractions containing chromatin bound proteins are shown. C. Rapid and reversible association of RECQ1 with damaged chromatin. HeLa cells were treated with 1 mM H₂O₂ for 20 min at 37° followed by incubation in H₂O₂-free medium for recovery, and subjected to sequential fractionation at indicated time points. Nuclear pellet fractions show relocalization of RECQ1, WRN, BLM and RecQ5β to damaged chromatin at different time during recovery. Levels of PCNA remain unchanged and serve as loading control. Untr., Untreated. D. Quantitation of chromatin enrichment of RecQ proteins at different time points following recovery from H₂O₂ (1 mM, 20 min) treatment. Western blots were quantitated using ImageJ. Signals were normalized using Histone (H3) and protein signal in the untreated was used to calculate fold enrichment following H₂O₂ treatment. Mean of relative fold enrichment and S.E.M. from three independent experiments are shown. E. H₂O₂-induced chromatin binding of RECQ1 and WRN is independent of PARP activity. HeLa cells grown in the presence or absence of ANI (100 µM) for 24 h, were mock-treated or treated with 1 mM H₂O₂ for 20 min. Cells were then sequentially fractionated and proteins from each fraction analyzed by Western blotting. The apparent decrease of RECQ1, WRN and XRCC1 proteins in the CSK fraction reflects the decrease of soluble nuclear proteins, which now associate tightly with the chromatin-enriched nuclear pellet. F. RECQ1-depleted cells show H₂O₂ -induced chromatin enrichment of PARP-1. PARP-1 was detected by Western blot in the chromatin fractions prepared from control or RECQ1-depleted cells that were either untreated or treated with 1 mM H₂O₂ for 20 min.