Formation of DDX1 IRIF upon exposure to γ-rays. (A to F) HeLa cells were grown on glass coverslips and exposed to 5 Gy of IR. One hour later, control cells (A to C) and irradiated cells (D to F) were fixed and double stained with anti-DDX1 antibody and anti-CstF64 (A and D), anti-Sm (B and E), and anti-PML (C and F) antibodies. Arrowheads indicate associations between DDX1 bodies and cleavage bodies (A), DDX1 bodies and CBs (B), and DDX1 bodies and PML bodies (C). Bar, 10 μm. (G) Whole-cell extracts from control HeLa cells and HeLa cells exposed to 5 Gy of IR were prepared. Fifty micrograms of protein was loaded into each lane. (H) Dose dependence of DDX1 IRIF. HeLa cells were exposed to 1, 2, 5, and 10 Gy and examined after 1 h of recovery. The number of foci per cell as a function of the radiation dose is indicated. (I) Time course of DDX1 IRIF formation. HeLa cells were exposed to 5 Gy of IR and examined at the time points indicated. The percentage of cells containing DDX1 IRIF at each time point is shown. Bars refer to standard errors of the means.

The formation of DDX1 IRIF requires functional ATM. (A) HeLa cells were pretreated with 20 μM wortmannin for 1 h and then subjected to 5 Gy of IR. Cells were immunostained after 1 h of recovery. (B, C, and D) DNA-PKcs-deficient M059J cells (B), ATM-deficient EBS cells (C), and ATM-expressing YZ5 cells (D) were treated with 5 Gy of IR. Cells were immunostained as described in the legend to panel A. (E) HeLa cells were treated with IR (5 Gy) and immunostained with anti-DDX1 and anti-pATM^Ser1981 antibodies 1 h post-IR. Bars, 10 μm.

DDX1 IRIF and transcription. (A) HeLa cells were incubated with 6 μg/ml of actinomycin D for 30 min and exposed to 5 Gy of IR. One hour later, cells were fixed and immunostained with anti-DDX1 and anti-γ-H2AX antibodies. (B) Ten hours after release from the thymidine block, HeLa cells were exposed to 5 Gy of IR and immunostained with anti-DDX1 and anti-γ-H2AX 30 min post-IR. Bars, 10 μm. DAPI, 4′,6-diamidino-2-phenylindole.

Characterization of DDX1 RNA-DNA-unwinding activity. (A) The RNA-DNA duplex R41(+)/D29(−)* was generated by the annealing of R41(+) with 5′-end ³²P-labeled D29(−) as described in Materials and Methods. Fifty femtomoles of duplexes was incubated with either 0.3 μg of recombinant DDX1 protein (lanes 3 to 8) or 0.1 U of RNase A (lanes 11 to 16) in the presence of 1 mM ATP, ATP-γ-S, ADP, or GTP as indicated. The R41(+)/D29(−)* duplex in lanes 1 and 9 and the D29(−)* oligonucleotide in lanes 2 and 10 were incubated in unwinding buffer without protein. Reaction mixtures containing heat-denatured DDX1 (boiled for 2 min) or RNase A (also boiled for 2 min) are indicated by the symbol Δ (lanes 8 and 16). All reaction mixtures were incubated for 20 min at 37°C prior to electrophoresis in a 12% polyacrylamide gel, followed by autoradiography. R, RNA; D, DNA; +, with; −, without. (B) Fifty femtomoles of 5′-end ³²P-labeled R29(−) was incubated with either 0.3 μg of recombinant DDX1 (lanes 2 to 7) or 0.1 U of RNase A (lanes 9 to 14). Mg²⁺ was omitted in lanes 6 and 13. Heat-denatured DDX1 and RNase A were used in lanes 7 and 14, respectively. (C to F) Reactions were carried out with 0.3 μg of DDX1 and D41(+)*/R29(−) (C), D29(+)*/R29(−) (D), D41(+)/R29(−)* (E), and D29(+)/R29(−)* (F), as described in the legend to panel A.

Colocalization of DDX1 IRIF with γ-H2AX foci. (A and B) Control HeLa cells (A) and HeLa cells treated with 5 Gy of IR (B) were examined 1 h after IR. Arrowheads indicate γ-H2AX foci that have no DDX1. (C) HeLa cells were treated with 80 μg/ml bleomycin for 2 h and subjected to immunostaining. (D) Normal GM38 fibroblasts were fixed 60 min after exposure to 5 Gy of IR and double stained with anti-DDX1 and anti-γ-H2AX antibodies. (E) An anti-DDX1 and anti-γ-H2AX antibody mixture was incubated with 1.5 μg/ml of a recombinant DDX1 peptide (amino acids 1 to 186) as a competitor for 4 h at room temperature. Double immunostaining of irradiated HeLa cells was then carried out as described in the legend to panel D. Bars, 10 μm.

Coimmunoprecipitation and phosphorylation of DDX1. (A) Whole-cell extracts from control and irradiated (IR) HeLa cells were incubated with either anti-DDX1 antibody or preimmune serum. The immunocomplexes were subjected to electrophoresis, transferred, and probed with the antibodies indicated. Six percent of the supernatant from each immunoprecipitation was loaded into the indicated lanes. IP, immunoprecipitation; IB, immunoblotting. (B) HeLa whole-cell extracts were immunoprecipitated with anti-ATM antibody. DDX1 was detected with anti-DDX1 antibody. (C) Endogenous ATM protein was immunoprecipitated from HeLa whole-cell extracts using anti-ATM antibody. The immunocomplex was incubated with 2 μg of recombinant DDX1 protein at 30°C for 20 min in the presence of [γ-³²P]ATP. Samples were divided and electrophoresed through two replicate SDS-10% PAGE gels. One gel (top panel) was transferred onto nitrocellulose, and the filter was exposed to film to detect phosphorylated DDX1. The second gel (bottom panel) was stained with Coomassie blue to allow the visualization of the recombinant DDX1 substrate used in each reaction. +, present; −, absent. (D) Endogenous ATM protein was immunoprecipitated from BT (ATM-proficient) and L3 (ATM-deficient) cells. The in vitro ATM kinase assay was performed as described in the legend to panel C. The phosphorylation of DDX1 was visualized by autoradiography. Immunoprecipitated ATM was detected with anti-ATM antibody using the same membrane. (E) Control HeLa cells and HeLa cells subjected to IR (5 Gy) were metabolically labeled with ³²P_i. Endogenous DDX1 protein from metabolically labeled cells was immunoprecipitated with anti-DDX1 antibody. The immunocomplex was fractionated in an SDS-PAGE gel and transferred, and phosphorylated DDX1 protein was visualized by autoradiography. The identity of DDX1 was confirmed by immunostaining the membrane with anti-DDX1 antibody. Where indicated, cells were pretreated with 100 μM wortmannin for 30 min before IR. Asterisks indicate coimmunoprecipitated unknown proteins that were also phosphorylated.

RNase H treatment dissociates DDX1 from IRIF. HeLa cells were treated with IR (5 Gy) and incubated at 37°C for 1 h to allow the IRIF to form. Cells were then permeabilized (using 2% Tween 20) (A) and treated with DNase I (B), RNase A (C), or RNase H (D). Cells were fixed and immunostained with anti-DDX1 and anti-γ-H2AX antibodies. Bars, 10 μm.

DDX1 unwinds RNA-RNA but not DNA-DNA duplexes. (A) The RNA-RNA duplex R41(+)/R29(−)* was generated by annealing R41(+) with 5′-end ³²P-labeled R29(−). Fifty femtomoles of the substrate was incubated with either 0.3 μg of recombinant DDX1 (lanes 3 to 6) or 0.1 U of RNase A (lanes 9 to 12) in the presence of 1 mM ATP, ADP, or GTP as indicated. The R29(−)* oligonucleotide in lanes 1 and 7 and the R41(+)/R29(−)* duplex in lanes 2 and 8 were incubated in unwinding buffer without protein. ATP was omitted from reaction mixtures loaded into lanes 6 and 12. All reactions were carried out for 20 min at 37°C prior to electrophoresis in a 12% polyacrylamide gel, followed by autoradiography. R, RNA; D, DNA; +, with; −, without. (B) Fifty femtomoles of D41(+)/D29(−)* was incubated with 0.3 μg of recombinant DDX1 (lanes 2 to 6) at 37°C for 20 min, and the mixture was then subjected to electrophoresis and autoradiography.