Human RECQ1 Is a DNA Damage Responsive Protein Required for Genotoxic Stress Resistance and Suppression of Sister Chromatid Exchanges
Figure 1
Nucleolar RECQ1 undergoes re-localization to chromatin in response to IR.
Indirect immunofluorescence was performed on HeLa cells that were either untreated or allowed to recover for 6 h from 10 Gy IR exposure as described in “Materials and Methods”. Panel A, Nucleolar localization of RECQ1 in untreated cells. Cells were co-immunostained with anti-RECQ1 and anti-nucleolin. The merged images show cells stained with RECQ1 (red) and nucleolin (green), with or without DAPI (blue). Panel B, Relocalization of RECQ1 to chromatin-bound foci following IR damage. After IR exposure, cells were stained for total RECQ1 (top panel) or in situ detergent extraction-resistant RECQ1 (middle and bottom panel). Merged images show RECQ1 (red) and DAPI (blue). Co-immunostaining of chromatin-bound RECQ1 and γ-H2AX (Panel B, bottom). Merged images show RECQ1 (red) and γ-H2AX (green), with or without DAPI (blue). Panel C, Schematic presentation of the protocol for sequential nuclear fractionation of lysates from U2OS cells either untreated or following 6 h recovery from 10 Gy IR exposure. Cytoplasmic and nucleoplasmic proteins were extracted by permeabilization with detergent, and the resulting nuclei were nuclease-digested and extracted with NH2SO4. Proteins of the supernatant (S) and pellet (P) fractions were resolved on 10% SDS-PAGE, and subsequently analyzed by Western blot for RECQ1, histone H4 (chromatin marker), and lamin B (nuclear matrix marker). Panel D, Following IR treatment, RECQ1 is enriched in the chromatin fraction (S4).