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Regulation and localization of the Bloom syndrome protein in response to DNA damage.

Bischof O, Kim SH, Irving J, Beresten S, Ellis NA, Campisi J - J. Cell Biol. (2001)

Bottom Line: DNA-damaging agents that cause double strand breaks and a G2 delay induced BLM by a p53- and ataxia-telangiectasia mutated independent mechanism.This induction depended on the G2 delay, because it failed to occur when G2 was prevented or bypassed.It coincided with the appearance of foci containing BLM, PML, hRAD51 and RP-A, which resembled ionizing radiation-induced foci.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA..

ABSTRACT
Bloom syndrome (BS) is an autosomal recessive disorder characterized by a high incidence of cancer and genomic instability. BLM, the protein defective in BS, is a RecQ-like helicase, presumed to function in DNA replication, recombination, or repair. BLM localizes to promyelocytic leukemia protein (PML) nuclear bodies and is expressed during late S and G2. We show, in normal human cells, that the recombination/repair proteins hRAD51 and replication protein (RP)-A assembled with BLM into a fraction of PML bodies during late S/G2. Biochemical experiments suggested that BLM resides in a nuclear matrix-bound complex in which association with hRAD51 may be direct. DNA-damaging agents that cause double strand breaks and a G2 delay induced BLM by a p53- and ataxia-telangiectasia mutated independent mechanism. This induction depended on the G2 delay, because it failed to occur when G2 was prevented or bypassed. It coincided with the appearance of foci containing BLM, PML, hRAD51 and RP-A, which resembled ionizing radiation-induced foci. After radiation, foci containing BLM and PML formed at sites of single-stranded DNA and presumptive repair in normal cells, but not in cells with defective PML. Our findings suggest that BLM is part of a dynamic nuclear matrix-based complex that requires PML and functions during G2 in undamaged cells and recombinational repair after DNA damage.

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BLM and PML localize to sites of putative DNA repair after IR. Proliferating WI-38 were labeled for two doublings with BrdU and X-irradiated (5 Gy) where indicated. 10–12 h after irradiation, the cells were fixed under nondenaturing conditions and immunostained for BrdU, BLM, and PML. Nuclei were visualized (DAPI) and fluorescent images were superimposed (MERGE) as described in the legend to Fig. 1. (a) BrdU staining in unirradiated cells. (b) BLM foci in unirradiated, BrdU-labeled cells. (c) BrdU and DAPI staining in irradiated cells. The BrdU and DAPI images were merged. (d) PARP integrity after irradiation. BrdU-labeled cells were left untreated (−IR), X-irradiated (+IR), or treated with Fas ligand (Fas; positive control for apoptosis). 10 h later, protein lysates were prepared and analyzed for PARP by Western blotting. (e–m) BLM and BrdU localization in irradiated cells. e–g show a cell with 80–90% colocalization of BLM and BrdU foci; 30–50% of cells with BrdU foci showed this staining pattern. h–m show cells in which BLM and BrdU colocalized to varying degrees. (n–p) PML and BrdU localization in irradiated cells. Bar, ∼10 μm.
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Figure 8: BLM and PML localize to sites of putative DNA repair after IR. Proliferating WI-38 were labeled for two doublings with BrdU and X-irradiated (5 Gy) where indicated. 10–12 h after irradiation, the cells were fixed under nondenaturing conditions and immunostained for BrdU, BLM, and PML. Nuclei were visualized (DAPI) and fluorescent images were superimposed (MERGE) as described in the legend to Fig. 1. (a) BrdU staining in unirradiated cells. (b) BLM foci in unirradiated, BrdU-labeled cells. (c) BrdU and DAPI staining in irradiated cells. The BrdU and DAPI images were merged. (d) PARP integrity after irradiation. BrdU-labeled cells were left untreated (−IR), X-irradiated (+IR), or treated with Fas ligand (Fas; positive control for apoptosis). 10 h later, protein lysates were prepared and analyzed for PARP by Western blotting. (e–m) BLM and BrdU localization in irradiated cells. e–g show a cell with 80–90% colocalization of BLM and BrdU foci; 30–50% of cells with BrdU foci showed this staining pattern. h–m show cells in which BLM and BrdU colocalized to varying degrees. (n–p) PML and BrdU localization in irradiated cells. Bar, ∼10 μm.

Mentions: Cells were grown for two doublings in the presence of BrdU. Proliferating BrdU-labeled cells, fixed and stained under nondenaturing conditions, showed no significant staining (Fig. 8 a), confirming that the antibody does not detect BrdU in duplex DNA. The same cells showed the expected heterogeneous pattern of BLM foci (Fig. 8 b), indicating that BrdU did not perturb BLM localization. When BrdU-labeled cells were X-irradiated (5 Gy), BrdU foci appeared (Fig. 8 c), peaking 8–10 h after IR. At this time, >50% of the cells had multiple BrdU foci. These foci were not due to apoptosis because there was no evidence of PARP degradation, which precedes apoptotic DNA fragmentation, for at least 10 h after IR (Fig. 8 d). Moreover, BrdU foci appeared to be a specific response to DSBs because UV induced few if any BrdU foci (not shown). Thus, in agreement with Raderschall et al. 1999, BrdU foci formed primarily in response to DNA DSBs, where they presumably identify sites of repair.


Regulation and localization of the Bloom syndrome protein in response to DNA damage.

Bischof O, Kim SH, Irving J, Beresten S, Ellis NA, Campisi J - J. Cell Biol. (2001)

BLM and PML localize to sites of putative DNA repair after IR. Proliferating WI-38 were labeled for two doublings with BrdU and X-irradiated (5 Gy) where indicated. 10–12 h after irradiation, the cells were fixed under nondenaturing conditions and immunostained for BrdU, BLM, and PML. Nuclei were visualized (DAPI) and fluorescent images were superimposed (MERGE) as described in the legend to Fig. 1. (a) BrdU staining in unirradiated cells. (b) BLM foci in unirradiated, BrdU-labeled cells. (c) BrdU and DAPI staining in irradiated cells. The BrdU and DAPI images were merged. (d) PARP integrity after irradiation. BrdU-labeled cells were left untreated (−IR), X-irradiated (+IR), or treated with Fas ligand (Fas; positive control for apoptosis). 10 h later, protein lysates were prepared and analyzed for PARP by Western blotting. (e–m) BLM and BrdU localization in irradiated cells. e–g show a cell with 80–90% colocalization of BLM and BrdU foci; 30–50% of cells with BrdU foci showed this staining pattern. h–m show cells in which BLM and BrdU colocalized to varying degrees. (n–p) PML and BrdU localization in irradiated cells. Bar, ∼10 μm.
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Figure 8: BLM and PML localize to sites of putative DNA repair after IR. Proliferating WI-38 were labeled for two doublings with BrdU and X-irradiated (5 Gy) where indicated. 10–12 h after irradiation, the cells were fixed under nondenaturing conditions and immunostained for BrdU, BLM, and PML. Nuclei were visualized (DAPI) and fluorescent images were superimposed (MERGE) as described in the legend to Fig. 1. (a) BrdU staining in unirradiated cells. (b) BLM foci in unirradiated, BrdU-labeled cells. (c) BrdU and DAPI staining in irradiated cells. The BrdU and DAPI images were merged. (d) PARP integrity after irradiation. BrdU-labeled cells were left untreated (−IR), X-irradiated (+IR), or treated with Fas ligand (Fas; positive control for apoptosis). 10 h later, protein lysates were prepared and analyzed for PARP by Western blotting. (e–m) BLM and BrdU localization in irradiated cells. e–g show a cell with 80–90% colocalization of BLM and BrdU foci; 30–50% of cells with BrdU foci showed this staining pattern. h–m show cells in which BLM and BrdU colocalized to varying degrees. (n–p) PML and BrdU localization in irradiated cells. Bar, ∼10 μm.
Mentions: Cells were grown for two doublings in the presence of BrdU. Proliferating BrdU-labeled cells, fixed and stained under nondenaturing conditions, showed no significant staining (Fig. 8 a), confirming that the antibody does not detect BrdU in duplex DNA. The same cells showed the expected heterogeneous pattern of BLM foci (Fig. 8 b), indicating that BrdU did not perturb BLM localization. When BrdU-labeled cells were X-irradiated (5 Gy), BrdU foci appeared (Fig. 8 c), peaking 8–10 h after IR. At this time, >50% of the cells had multiple BrdU foci. These foci were not due to apoptosis because there was no evidence of PARP degradation, which precedes apoptotic DNA fragmentation, for at least 10 h after IR (Fig. 8 d). Moreover, BrdU foci appeared to be a specific response to DSBs because UV induced few if any BrdU foci (not shown). Thus, in agreement with Raderschall et al. 1999, BrdU foci formed primarily in response to DNA DSBs, where they presumably identify sites of repair.

Bottom Line: DNA-damaging agents that cause double strand breaks and a G2 delay induced BLM by a p53- and ataxia-telangiectasia mutated independent mechanism.This induction depended on the G2 delay, because it failed to occur when G2 was prevented or bypassed.It coincided with the appearance of foci containing BLM, PML, hRAD51 and RP-A, which resembled ionizing radiation-induced foci.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA..

ABSTRACT
Bloom syndrome (BS) is an autosomal recessive disorder characterized by a high incidence of cancer and genomic instability. BLM, the protein defective in BS, is a RecQ-like helicase, presumed to function in DNA replication, recombination, or repair. BLM localizes to promyelocytic leukemia protein (PML) nuclear bodies and is expressed during late S and G2. We show, in normal human cells, that the recombination/repair proteins hRAD51 and replication protein (RP)-A assembled with BLM into a fraction of PML bodies during late S/G2. Biochemical experiments suggested that BLM resides in a nuclear matrix-bound complex in which association with hRAD51 may be direct. DNA-damaging agents that cause double strand breaks and a G2 delay induced BLM by a p53- and ataxia-telangiectasia mutated independent mechanism. This induction depended on the G2 delay, because it failed to occur when G2 was prevented or bypassed. It coincided with the appearance of foci containing BLM, PML, hRAD51 and RP-A, which resembled ionizing radiation-induced foci. After radiation, foci containing BLM and PML formed at sites of single-stranded DNA and presumptive repair in normal cells, but not in cells with defective PML. Our findings suggest that BLM is part of a dynamic nuclear matrix-based complex that requires PML and functions during G2 in undamaged cells and recombinational repair after DNA damage.

Show MeSH
Related in: MedlinePlus