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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 localizes with hRAD51, RP-A, and PML after IR. Proliferating WI-38, BS HG2654, and NB4 cells were X-irradiated (5 Gy) where indicated. 10 h after irradiation, the cells were immunostained for BLM, PML, hRAD51, or RP-A; nuclei were visualized (DAPI); and fluorescent images were superimposed (MERGE) as described in the legend to Fig. 1. (a–d) BLM and hRAD51 localization in irradiated WI-38 cells. (e–h) BLM and RP-A localization in irradiated WI-38 cells. (i–l) BLM and PML localization in irradiated WI-38 cells. (m–o) hRAD51 localization in irradiated BS cells. (p–s) hRAD51 and BLM localization in unirradiated (−IR) and irradiated (+IR) NB4 cells. Bars, ∼10 μm.
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Figure 7: BLM localizes with hRAD51, RP-A, and PML after IR. Proliferating WI-38, BS HG2654, and NB4 cells were X-irradiated (5 Gy) where indicated. 10 h after irradiation, the cells were immunostained for BLM, PML, hRAD51, or RP-A; nuclei were visualized (DAPI); and fluorescent images were superimposed (MERGE) as described in the legend to Fig. 1. (a–d) BLM and hRAD51 localization in irradiated WI-38 cells. (e–h) BLM and RP-A localization in irradiated WI-38 cells. (i–l) BLM and PML localization in irradiated WI-38 cells. (m–o) hRAD51 localization in irradiated BS cells. (p–s) hRAD51 and BLM localization in unirradiated (−IR) and irradiated (+IR) NB4 cells. Bars, ∼10 μm.

Mentions: Although most (60–90%) PML NBs stained for BLM during late S/G2, many were devoid of BLM at other cell cycle stages. By contrast, most (80–90%) BLM foci stained for PML, regardless of cell cycle position. The few BLM foci that apparently lacked PML may indicate rare BLM localization outside PML NBs, or failure of the antibody to recognize PML in all NBs. Whatever the case, the majority of BLM colocalized with PML in human HCA2 normal fibroblasts, HT1080 fibrosarcoma, SAOS osteosarcoma, and VA-13 SV40-transformed fibroblasts (not shown). The exception was NB4 cells, which express a dominant negative form of PML and show abnormal PML organization into small aggregates or microspeckles (de The et al. 1991; Mu et al. 1994). As reported by Zhong et al. 1999, BLM showed mostly diffuse staining in NB4 nuclei (see Fig. 7 r), suggesting that PML is important, if not essential, for BLM focus formation.


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 localizes with hRAD51, RP-A, and PML after IR. Proliferating WI-38, BS HG2654, and NB4 cells were X-irradiated (5 Gy) where indicated. 10 h after irradiation, the cells were immunostained for BLM, PML, hRAD51, or RP-A; nuclei were visualized (DAPI); and fluorescent images were superimposed (MERGE) as described in the legend to Fig. 1. (a–d) BLM and hRAD51 localization in irradiated WI-38 cells. (e–h) BLM and RP-A localization in irradiated WI-38 cells. (i–l) BLM and PML localization in irradiated WI-38 cells. (m–o) hRAD51 localization in irradiated BS cells. (p–s) hRAD51 and BLM localization in unirradiated (−IR) and irradiated (+IR) NB4 cells. Bars, ∼10 μm.
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Figure 7: BLM localizes with hRAD51, RP-A, and PML after IR. Proliferating WI-38, BS HG2654, and NB4 cells were X-irradiated (5 Gy) where indicated. 10 h after irradiation, the cells were immunostained for BLM, PML, hRAD51, or RP-A; nuclei were visualized (DAPI); and fluorescent images were superimposed (MERGE) as described in the legend to Fig. 1. (a–d) BLM and hRAD51 localization in irradiated WI-38 cells. (e–h) BLM and RP-A localization in irradiated WI-38 cells. (i–l) BLM and PML localization in irradiated WI-38 cells. (m–o) hRAD51 localization in irradiated BS cells. (p–s) hRAD51 and BLM localization in unirradiated (−IR) and irradiated (+IR) NB4 cells. Bars, ∼10 μm.
Mentions: Although most (60–90%) PML NBs stained for BLM during late S/G2, many were devoid of BLM at other cell cycle stages. By contrast, most (80–90%) BLM foci stained for PML, regardless of cell cycle position. The few BLM foci that apparently lacked PML may indicate rare BLM localization outside PML NBs, or failure of the antibody to recognize PML in all NBs. Whatever the case, the majority of BLM colocalized with PML in human HCA2 normal fibroblasts, HT1080 fibrosarcoma, SAOS osteosarcoma, and VA-13 SV40-transformed fibroblasts (not shown). The exception was NB4 cells, which express a dominant negative form of PML and show abnormal PML organization into small aggregates or microspeckles (de The et al. 1991; Mu et al. 1994). As reported by Zhong et al. 1999, BLM showed mostly diffuse staining in NB4 nuclei (see Fig. 7 r), suggesting that PML is important, if not essential, for BLM focus formation.

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