DEAD Box 1 Facilitates Removal of RNA and Homologous Recombination at DNA Double-Strand Breaks

Abstract

Although RNA and RNA-binding proteins have been linked to double-strand breaks (DSBs), little is known regarding their roles in the cellular response to DSBs and, if any, in the repair process. Here, we provide direct evidence for the presence of RNA-DNA hybrids at DSBs and suggest that binding of RNA to DNA at DSBs may impact repair efficiency. Our data indicate that the RNA-unwinding protein DEAD box 1 (DDX1) is required for efficient DSB repair and cell survival after ionizing radiation (IR), with depletion of DDX1 resulting in reduced DSB repair by homologous recombination (HR). While DDX1 is not essential for end resection, a key step in homology-directed DSB repair, DDX1 is required for maintenance of the single-stranded DNA once generated by end resection. We show that transcription deregulation has a significant effect on DSB repair by HR in DDX1-depleted cells and that RNA-DNA duplexes are elevated at DSBs in DDX1-depleted cells. Based on our combined data, we propose a role for DDX1 in resolving RNA-DNA structures that accumulate at DSBs located at sites of active transcription. Our findings point to a previously uncharacterized requirement for clearing RNA at DSBs for efficient repair by HR.

FIG 1

DDX1 promotes cell survival and DSB repair in irradiated cells. (A) U2OS cells transfected with scrambled siRNAs (control) or siRNAs targeting DDX1 (siRNA1 [si1] and siRNA2 [si2]) were exposed to the indicated doses of IR. Cell survival was measured by colony formation assay. Inset, Western blot of U2OS cells transfected with scrambled siRNAs (control) or DDX1 siRNAs. (B) Statistical analysis of average numbers of γ-H2AX foci in control and DDX1 knockdown cells at 0.5, 1, 4, and 24 h after 3 Gy IR. (C) Control and DDX1 knockdown U2OS cells were exposed to 3 Gy IR. Immunostaining with anti-γ-H2AX antibody was performed 4 h post-IR. (D) Left, U2OS and U2OS HA-mDDX1 cells that stably express siRNA-resistant DDX1 were transfected with scrambled (−) or DDX1 (+) siRNA. Numbers of γ-H2AX foci were analyzed 4 h after 3 Gy IR. For U2OS HA-mDDX1 cells, only cells that express HA-mDDX1 (positive for HA staining) were analyzed. Right, Western blot of U2OS HA-mDDX1 cell lysates that were transfected with scrambled siRNAs (−) or DDX1 siRNA (+). The asterisk indicates HA-tagged DDX1 that is resistant to DDX1 siRNA. The arrowhead points to endogenous DDX1. (E) U2OS cells were immunostained with the indicated antibodies 4 h after 3 Gy IR. Cells in different phases of the cell cycle were distinguished by CENP-F intensities: weak, medium, and strong CENP-F immunostaining indicates cells in G₁, S, and G₂ phases, respectively. (F) Numbers of γ-H2AX foci in control and DDX1 knockdown U2OS cells were analyzed 4 h after 3 Gy IR. Cells in G₁ and G₂ phases were distinguished by CENP-F immunostaining intensities. Ratios of γ-H2AX foci in control versus DDX1 knockdown cells in G₁ and G₂ phases are depicted in the graphs. For all samples, n = 3; error bars are standard errors of the means (SEM). P values were calculated using the two-sided Student t test. Scale bars, 10 μm.

FIG 2

DDX1 facilitates HR-mediated DSB repair. (A) U2OS DR-GFP cells were transfected with scrambled siRNAs (control), DDX1 siRNA (si1 and si2), or RAD51 siRNA. After 72 h, cells were transfected with the same siRNAs along with an I-SceI expression construct by electroporation. GFP-positive cells were analyzed by flow cytometry. Relative HR efficiency was calculated by comparing percentages of GFP-positive cells in DDX1 siRNA- or RAD51 siRNA-treated cells versus control transfectants. (B) U2OS DR-GFP and U2OS DR-GFP HA-mDDX1 cells that stably express siRNA-resistant DDX1 were transfected with scrambled siRNAs (−) or DDX1 si1 (+). Relative HR efficiency was measured as described in for panel A. (C) Control and DDX1-depleted U2OS cells were treated with AG14361 at the indicated concentrations for 24 h. Cell survival was measured using the clonogenic assay. (D) Measurement of NHEJ efficiency in control and DDX1-depleted U2OS EJ5-GFP cells. Relative repair efficiency was calculated as described in for panel A. (E) Control and DDX1-depleted U2OS cells were exposed to 3 Gy IR. Cells were immunostained with anti-RAD51 antibody at 2 h post-IR. Bar, 10 μm. (F) U2OS cells and U2OS HA-mDDX1 cells that stably express siRNA-resistant DDX1 were transfected with scrambled (−) or DDX1 (+) siRNA. Numbers of RAD51 foci were analyzed 2 h after 3 Gy IR. For U2OS HA-mDDX1 cells, only cells that express HA-mDDX1 (positive for HA staining) were analyzed. (G) U2OS cells were transfected with scrambled or DDX1 siRNAs. Forty micrograms of lysates was resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Asterisk, residual actin signal. (H) U2OS cells were synchronized using a double thymidine block method. The numbers of RAD51 foci in control and DDX1-depleted cells were analyzed 2 h after 3 Gy treatment in S phase and G₂ phase. For all samples, n = 3, except for panel F, where n = 2; error bars are SEM. P values were calculated using Fisher's exact test for panels A and H and Student's t test for panel F.

FIG 3

DDX1 depletion results in decreased single-stranded DNA post-IR treatment. (A) U2OS cells were transfected with scrambled siRNA (control) or DDX1 si1. Cells were exposed to 5 Gy IR, extracted, and immunostained with anti-RPA antibody at 2 h post-IR. (B) U2OS and U2OS HA-mDDX1 cells were transfected with scrambled (−) or DDX1 (+) siRNA. RPA foci were analyzed 2 h after 5 Gy IR. For U2OS HA-mDDX1 cells, only cells that express HA-mDDX1 (positive for HA staining) were analyzed. (C) U2OS cells were synchronized using the double thymidine block method. Numbers of RPA foci were analyzed in S phase and G₂ phase of control or DDX1-depleted cells. (D) Control and DDX1-depleted U2OS cells were incubated with 10 μM BrdU for 24 h and then exposed to 5 Gy IR. Nondenaturing BrdU staining was performed at 3 h post-IR. Note that BrdU also stains mitochondrial DNA in the cytoplasm. The contour of the nuclei is indicated by the dotted lines. (E) Percentages of untreated and IR-treated control and DDX1-depleted cells that are positive for nuclear BrdU. (F) Control and DDX1-depleted U2OS cells were treated with 15 Gy IR and harvested 2 h later. Nuclear extracts were resolved by SDS-PAGE and immunostained with the indicated antibodies. (G and H) Control and DDX1-depleted U2OS cells were treated with 1 μM CPT for 1 h (G) or 30 min (H). Cells were processed as described above. The arrowhead points to phosphorylated RPA. Scale bars, 10 μm; error bars, SEM; n = 3 for all samples. P values were calculated using the two-sided Student t test (B) or Fisher's exact test (C and E).

FIG 4

DDX1 depletion has little effect on end resection. (A) Control and DDX1-depleted U2OS cells were microirradiated. Cells were immunostained with anti-γ-H2AX, anti-cyclin A, and anti-CtIP antibodies at 1 h postirradiation. Note that under these conditions, CtIP recruitment occurs in S/G₂-phase cells (indicated by positive nuclear staining of cyclin A, shown by arrows) but not in G₁-phase cells (negative nuclear cyclin A staining, shown by arrowheads). (B) Percentage of γ-H2AX stripes with CtIP recruitment in control and DDX1 knockdown cells treated as described for panel A. (C) Control and DDX1-depleted U2OS cells were treated with 2 Gy IR and immunostained with anti-γ-H2AX and anti-pEXO1 antibodies at 1 h post-IR. (D) Percentage of cells containing pEXO1 foci and number of foci per individual cell at 1 h (left) or 3 h (right) post-IR. P values were calculated using the two-sided Student t test for panel B and the chi-square test with Yates' correction for panel D. Scale bars, 20 μm; error bars, SEM; n = 3.

FIG 5

Formation of IR-induced DDX1 foci is dependent on efficient end resection. (A) Control and CtIP-depleted HeLa cells were immunostained with anti-γ-H2AX and anti-DDX1 antibodies at 3 h after treatment with 5 Gy IR. Scale bar, 20 μm. (B) Cells were treated as described for panel A. The average numbers of IR-induced DDX1 foci in control, CtIP knockdown, or RNF138 knockdown cells were analyzed (n = 3). P values were calculated using Fisher's exact test. Error bars, SEM. (C) HeLa cells were transfected with scrambled (control), CtIP, or RNF138 siRNAs. Seventy-two hours later, cells were harvested. Forty micrograms of whole-cell lysates was separated by SDS-PAGE and immunoblotted with the indicated antibodies.

FIG 6

Effects of RNA transcription on DDX1 recruitment/retention at DNA damage sites and DDX1-mediated HR repair. (A) HeLa cells were treated with 100 μM cordycepin for 2 h (bottom panel) or mock treated (top panel). Cells were then exposed to 5 Gy IR and immunostained at 1 h post-IR. (B) HeLa cells were treated with cordycepin or mock treated as described above. Cells were then incubated with 1 μM camptothecin for 1 h, followed by immunostaining. Scale bars, 20 μm. (C) HeLa cells were treated as described for panel B. Forty micrograms of lysates was resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (D) A schematic representation of sense RNA and antisense RNA and their locations relative to the I-SceI cut site in the DR-GFP reporter. The bent arrow indicates the GFP transcription orientation. (E) Empty vector or constructs expressing sense RNA or antisense RNA were transfected into control and DDX1-depleted U2OS DR-GFP cells. Relative HR efficiency was measured as described for Fig. 2A (n = 3). Error bars, SEM. P values were calculated using Student's t test.

FIG 7

DDX1 promotes clearance of DNA-RNA duplexes formed at the I-SceI-induced DSBs. (A) A schematic illustration showing the position of the I-SceI-induced DSB in the DR-GFP reporter and the three sites (P1 to P3) targeted for analysis by qPCR. (B) U2OS DR-GFP cells were transfected with scrambled siRNA (control) or DDX1 siRNA (DDX1 si1). Cells were then transfected with the HA-ER-I-SceI construct, and DSBs at the I-SceI site were induced with 5 μM tamoxifen for 4 h. Genomic DNA (50 ng) was amplified by semiquantitative PCR with primers flanking the I-SceI cut site (labeled P1 in panel A) (upper panel) or GAPDH-specific primers (bottom panel). (C) qPCR analysis of the region spanning the I-Sce I cut site (P1 site) in cells described for panel B. Signal intensities were normalized against a GAPDH control. (D and E) DRIP analysis was carried out using the S9.6 antibody and nucleic acid purified from control (scrambled siRNA-transfected) and DDX1-depleted U2OS DR-GFP cells with DSBs induced at I-SceI sites. qPCR was performed using primers specific to sites P2 (D) and P3 (E). Where indicated, samples were treated with RNase H prior to DRIP analysis. Signal intensities were normalized against input DNA as well as the SNRPN negative control. (F) DRIP-qPCR analysis was performed as described above. Where indicated, samples were treated with RNase A prior to DRIP. Relative increases in the qPCR signal were calculated by comparing the ratio of the qPCR signal in DDX1-depleted versus control cells treated with RNase A with the ratio of the qPCR signal in DDX1-depleted versus control cells without RNase A treatment. (G) U2OS DR-GFP cells stably expressing HA-mDDX1 were transfected with scrambled siRNA (−) or DDX1 si1 (+). DRIP-qPCR analysis was performed as described for panels D and E. P values were calculated using the Mann-Whitney test; n = 3 for all cases.

FIG 8

DDX1 promotes RNA clearance at I-SceI-induced DSBs. (A) Three possible scenarios at I-SceI-induced DSBs. The positions of primers (upstream region) are indicated by the green arrows. Note that RNA molecules can be either long and continuous (as shown) or short and discontinuous. (B) DSBs at I-SceI sites were induced by tamoxifen in control or DDX1-depleted U2OS DR-GFP cells. One microgram of nucleic acid was digested with DSN alone or RNase H followed by DSN. Digested DNA (250 ng) was PCR amplified using primers specific to the upstream region of the I-SceI cut site (lanes 1 to 4) or GAPDH primers (lanes 5 to 8). For comparison, 50 ng of intact DNA from control cells (transfected with scrambled siRNA and ER-I-SceI expression vector and induced by tamoxifen) was PCR amplified using GAPDH primers (lane 9). (C) Average intensity of PCR products in panel B from four independent experiments. The P value was calculated using Student's t test. (D) Control or DDX1-depleted U2OS DR-GFP cells were transfected with an ER expression construct, followed by tamoxifen induction. PCR was performed as described for panel B. (E) Model of the proposed role for DDX1 in HR. See the text for details.