AT cells show a slight delay in 53BP1 focus formation in response to X-ray irradiation. AT cells (A to C) or HT1080 cells (D to F) were either untreated (A and D) or exposed to X rays, fixed at 1 h (B and E) or at 3 h (C and F) postirradiation, and stained for 53BP1.

XL53BP1 focus formation is diminished or delayed in the presence of caffeine or wortmannin, respectively. Cells were treated as described for Fig. 6. XTC-2 cells were exposed to X rays either in the absence of any drugs (A and B) or in the presence of 16 mM caffeine (C and D) or 23 μM wortmannin (E and F) and fixed at 30 min (A, C, and E) or at 3 h (B, D, and F) postirradiation for XL53BP1 stain.

Neither inhibitors of transcription nor translation blocks the assembly of XL53BP1. XTC-2 cells were irradiated with 5 Gy of γ rays in the presence of 50 μg of cycloheximide (Cyh) per ml (C) or 25 μg of actinomycin D (Act) per ml (D). (A) Nonirradiated control. (B) Cells irradiated without the inhibitors. (E) the amount of XL53BP1 did not increase after γ-ray irradiation. XTC-2 cells were exposed to 5 or 15 Gy of γ rays, and at 2 h after irradiation, cell extracts were prepared for immunoblotting with anti-XL53BP1 antibody. Each slot was loaded with the same amount of proteins.

(A) γ-Ray-dose-dependent increase of XL53BP1 focal number. The average number of foci per nucleus was plotted versus radiation dose. Error bars indicate standard deviations. (B) Recovery of the XL53BP1 focal phenotype after γ-ray irradiation. XTC-2 cells were either untreated (stippled bars) or irradiated with 5 Gy of γ rays (black bars) and fixed for XL53BP1 staining at 2 h or at 24 h postirradiation. Optical sections were recorded by confocal microscope. The number of XL53BP1 foci per nucleus was counted in 150 cells for each sample. At 2 h postirradiation, the nuclei had 10.9 ± 3.2 XL53BP1 foci (upper graph, black bars). At 24 h postirradiation, the average focus number per nucleus decreased to 1.99 ± 2.15 (lower graph, black bars), which was equivalent to that of the untreated control cells (stippled bars).

(A) Verification of antibodies against 53BP1 and immunoprecipitation. Cell lysates were prepared from Xenopus cells (XTC-2), (lanes 1 and 2) or from HeLa cells (lanes 3 and 4), run on an SDS-polyacrylamide gel, and subjected to immunoblotting. Affinity-purified anti-XL53BP1 (Xenopus cells; lane 1) or anti-HS53BP1 (human; lane 4) antibodies react with single proteins in the extracts. Preimmune sera were used as negative controls (lanes 2 and 3). Lane 6, fluorography of immunoprecipitate (IP) with anti-HS53BP1 antibody from ³⁵S-labeled HeLa cell extracts lane 5, immunoprecipitation with preimmune serum control. (B) Gel filtration of HeLa cell extracts. The extract (Lo) was loaded on a Superdex 200 (10/30) column, and the fractions were analyzed by immunoblotting with anti-HS53BP1 antibodies. Positions where the molecular mass markers were eluted are indicated below the gel in kilodaltons.

XL53BP1 localizes to nuclear foci, which increase in number after DNA damage. Xenopus XTC-2 cells were immunostained with anti-XL53BP1 antibodies. (A) In control cells, there were two types of XL53BP1 populations. One was homogeneously distributed over chromatin, and the other was associated with large foci. (B) Staining with preimmune serum showed very little background. (C to E) After exposure to γ rays, XL53BP1 formed increased numbers of smaller foci dispersed throughout the nucleus. γ-Ray dosages were 1.5 Gy (C), 7.5 Gy (D), and 20 Gy (E). (F to I) XL53BP1 foci were also induced by various DNA-damaging agents. Cells were subjected to 3.3 μg of VM26 per ml (F), 60 μg of mitomycin C per ml (G), 300 J of UV per m² (H), and 10 mM hydroxyurea (HU) (I). Cells were fixed for staining at 2 h postirradiation or at 16 h after addition of the chemicals.

Colocalization of 53BP1 foci and Mre11 foci in human HT1080 cells. HT1080 cells untreated (A, C, and E) or exposed to 84 Gy of X rays (X) (B, D, and F) were fixed at 15 h postirradiation and double stained with Cy5 for Mre11 (C and D; pseudo-colored in red) and with FITC for 53BP1 (A and B; green). (E and F) Merged images of panels A and C and of B and D, respectively. Colocalization of green foci and red foci yields yellow signals. The cell marked by an arrowhead in panels B and D had 53BP1 foci but not discrete Mre11 foci. The cells marked by asterisks in panels C and D were in mitosis, and 53BP1 and Mre11 were dissociated from mitotic chromosomes and spread throughout the cytoplasm. (G) Kinetics of 53BP1 and Mre11 focal formation. HT1080 cells were either untreated (0 h) or irradiated with X rays, fixed at the indicated times postirradiation, and stained independently for either 53BP1 or Mre11. Optical sections were recorded by confocal microscope. The number of 53BP1 foci (upper graph) or Mre11 (lower graph) foci per nucleus was counted in 50 cells for each sample. Nuclei were categorized as having 0 to 5 foci (blue bars), 6 to 30 foci (green bars), 31 to 60 foci (yellow bars), 61 to 90 foci (red bars), and >91 foci (brown bars) at each time point. The y axis indicates the percentage of each category at each time point. For Mre11, only large, well-distinguished foci were scored (e.g., the nucleus marked with an arrowhead in panel D was scored as negative for Mre11 foci). Thus, the number of Mre11 foci may have been underestimated. Note that 53BP1 foci form early at 30 min postirradiation, when Mre11 foci were not observed at all.

Phosphorylation of XL53BP1 in cells irradiated with X rays. (A) XL53BP1 was immunoprecipitated from XTC-2 cells either untreated (lanes 1 and 3; − X ray) or exposed to X rays (lanes 2 and 4; + X ray) at 2 h postirradiation. Half of the samples were treated with protein phosphatase (lanes 1 and 4; + PPase). The proteins were run on a gel and immunoblotted with anti-XL53BP1 antibodies. XL53BP1 of irradiated cells migrated more slowly than that of nonirradiated cells (lanes 2). This retardation was lost if the sample was phosphatased (lane 4). Lanes 5 and 6 indicate that the band retardation was already seen at 30 min postirradiation. (B) Caffeine blocks the phosphorylation of XL53BP1. XTC-2 cells were either untreated (lanes 7 to and 9) or exposed to X rays (lanes 1 to 6) in the absence of drugs (lanes 1, 4, and 7) or in the presence of 16 mM caffeine (lanes 2, 5, and 8) or 23 μM wortmannin (lanes 3, 6, and 9). Extracts were prepared from irradiated cells at 30 min (lanes 1, 2, and 3) and at 3 h (lanes 4 to 6) post-X-ray exposure. The extent of the band retardation was significantly reduced in the presence of caffeine (e.g., compare lanes 4 and 5). Wortmannin had a similar effect but with lower efficiency (lane 6). The same amount of protein was loaded per slot. The lower bands were degradation products of XL53BP1. (C) AT cells show a reduction in 53BP1 phosphorylation in response to X-ray irradiation. Extracts were prepared from untreated cells (lanes 1, 5, and 9) or from cells irradiated with X rays, at 30 min (lanes 2 and 8), at 1 h (lanes 3 and 7), and at 3 h (lanes 4 and 6) postirradiation. In control ATM⁺ HT1080 cells (lanes 1 to 5), 53BP1 showed slower migration after X-ray irradiation (lanes 2 to 4). In AT cells that are ATM kinase defective (lanes 6 to 9), the retardation was greatly reduced and the 53BP1 band showed only a slight retardation at 1 and 3 h compared with that of the untreated control (compare lanes 6 and 7 to 9). 53BP1 from the untreated HT1080 cells ran slightly more slowly than that from AT cells for unknown reasons. Samples were loaded in an order to see the shifted and the nonshifted bands next to each other.