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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 associates with the nuclear matrix and hRAD51. Nuclear matrices were prepared from proliferating WI-38 cells by high salt extraction (NaCl), or low salt extraction and amine modification (NH2SO4). After extraction, 30 μg of protein was analyzed from whole cell (Total), nuclear (Nuclear), and cytoplasmic (Cytosol) lysates, and the supernatants (S) and nuclear matrix pellets (P). Proteins were analyzed for BLM, α-tubulin (Tubulin; cytosolic marker), lamin B (nuclear matrix marker), PARP, and Ku70 (DNA-associated) by Western blotting. (a–b) Results of two independent fractionations. (c) Recombinant GST-BLM. GST-BLM was produced by baculovirus in insect cells. Nuclear proteins from infected cells were bound to glutathione-Sepharose, the resin was transferred to a column, and bound proteins (200 ng) were eluted and analyzed by silver-stained SDS-PAGE. (d) hRAD51 associates with BLM. GST or GST-BLM, bound to glutathione-Sepharose beads, were incubated with SAOS-2 nuclear lysates (Input) and transferred to a column. After washing, proteins were eluted from the columns (GST, Eluted; GST-BLM, Eluted), and proteins resistant to elution were released by boiling in SDS-PAGE sample buffer (GST, Boiled; GST-BLM, Boiled). Input, eluted, and released proteins were analyzed for BLM, hRAD51, PARP, and Ku70 by Western blotting. (e) hRAD51 coimmunoprecipitates with BLM from nuclear lysates. Nuclear lysates from SOAS-2 cells (Input) were precleared and immunoprecipitated with nonspecific (IgG) or anti-hRAD51 (α-RAD51) antibody, and the immunoprecipitates were analyzed for BLM and hRAD51 by SDS-PAGE and Western blotting, as described in Materials and Methods.
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Figure 3: BLM associates with the nuclear matrix and hRAD51. Nuclear matrices were prepared from proliferating WI-38 cells by high salt extraction (NaCl), or low salt extraction and amine modification (NH2SO4). After extraction, 30 μg of protein was analyzed from whole cell (Total), nuclear (Nuclear), and cytoplasmic (Cytosol) lysates, and the supernatants (S) and nuclear matrix pellets (P). Proteins were analyzed for BLM, α-tubulin (Tubulin; cytosolic marker), lamin B (nuclear matrix marker), PARP, and Ku70 (DNA-associated) by Western blotting. (a–b) Results of two independent fractionations. (c) Recombinant GST-BLM. GST-BLM was produced by baculovirus in insect cells. Nuclear proteins from infected cells were bound to glutathione-Sepharose, the resin was transferred to a column, and bound proteins (200 ng) were eluted and analyzed by silver-stained SDS-PAGE. (d) hRAD51 associates with BLM. GST or GST-BLM, bound to glutathione-Sepharose beads, were incubated with SAOS-2 nuclear lysates (Input) and transferred to a column. After washing, proteins were eluted from the columns (GST, Eluted; GST-BLM, Eluted), and proteins resistant to elution were released by boiling in SDS-PAGE sample buffer (GST, Boiled; GST-BLM, Boiled). Input, eluted, and released proteins were analyzed for BLM, hRAD51, PARP, and Ku70 by Western blotting. (e) hRAD51 coimmunoprecipitates with BLM from nuclear lysates. Nuclear lysates from SOAS-2 cells (Input) were precleared and immunoprecipitated with nonspecific (IgG) or anti-hRAD51 (α-RAD51) antibody, and the immunoprecipitates were analyzed for BLM and hRAD51 by SDS-PAGE and Western blotting, as described in Materials and Methods.

Mentions: To determine how BLM associates with the PML NB, we prepared nuclear matrices from proliferating WI-38 cells using either of two methods: (a) standard high-salt extraction of nuclei, or (b) extraction at lower ionic strength followed by amine modification, which avoids the potential artifact of nonspecific salt precipitation (Wan et al. 1999). Proteins in the matrix and other fractions were analyzed by Western blotting. Regardless of the preparation method, BLM was found predominantly in the nuclear matrix fraction, cofractionating with the nuclear matrix marker protein lamin B (Spector 1993; Lamond and Earnshaw 1998; Wan et al. 1999; Fig. 3, a and b). BLM also fractionated with the nuclear matrix in VA-13 and HT1080 tumor cells (not shown).


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 associates with the nuclear matrix and hRAD51. Nuclear matrices were prepared from proliferating WI-38 cells by high salt extraction (NaCl), or low salt extraction and amine modification (NH2SO4). After extraction, 30 μg of protein was analyzed from whole cell (Total), nuclear (Nuclear), and cytoplasmic (Cytosol) lysates, and the supernatants (S) and nuclear matrix pellets (P). Proteins were analyzed for BLM, α-tubulin (Tubulin; cytosolic marker), lamin B (nuclear matrix marker), PARP, and Ku70 (DNA-associated) by Western blotting. (a–b) Results of two independent fractionations. (c) Recombinant GST-BLM. GST-BLM was produced by baculovirus in insect cells. Nuclear proteins from infected cells were bound to glutathione-Sepharose, the resin was transferred to a column, and bound proteins (200 ng) were eluted and analyzed by silver-stained SDS-PAGE. (d) hRAD51 associates with BLM. GST or GST-BLM, bound to glutathione-Sepharose beads, were incubated with SAOS-2 nuclear lysates (Input) and transferred to a column. After washing, proteins were eluted from the columns (GST, Eluted; GST-BLM, Eluted), and proteins resistant to elution were released by boiling in SDS-PAGE sample buffer (GST, Boiled; GST-BLM, Boiled). Input, eluted, and released proteins were analyzed for BLM, hRAD51, PARP, and Ku70 by Western blotting. (e) hRAD51 coimmunoprecipitates with BLM from nuclear lysates. Nuclear lysates from SOAS-2 cells (Input) were precleared and immunoprecipitated with nonspecific (IgG) or anti-hRAD51 (α-RAD51) antibody, and the immunoprecipitates were analyzed for BLM and hRAD51 by SDS-PAGE and Western blotting, as described in Materials and Methods.
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Related In: Results  -  Collection

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Figure 3: BLM associates with the nuclear matrix and hRAD51. Nuclear matrices were prepared from proliferating WI-38 cells by high salt extraction (NaCl), or low salt extraction and amine modification (NH2SO4). After extraction, 30 μg of protein was analyzed from whole cell (Total), nuclear (Nuclear), and cytoplasmic (Cytosol) lysates, and the supernatants (S) and nuclear matrix pellets (P). Proteins were analyzed for BLM, α-tubulin (Tubulin; cytosolic marker), lamin B (nuclear matrix marker), PARP, and Ku70 (DNA-associated) by Western blotting. (a–b) Results of two independent fractionations. (c) Recombinant GST-BLM. GST-BLM was produced by baculovirus in insect cells. Nuclear proteins from infected cells were bound to glutathione-Sepharose, the resin was transferred to a column, and bound proteins (200 ng) were eluted and analyzed by silver-stained SDS-PAGE. (d) hRAD51 associates with BLM. GST or GST-BLM, bound to glutathione-Sepharose beads, were incubated with SAOS-2 nuclear lysates (Input) and transferred to a column. After washing, proteins were eluted from the columns (GST, Eluted; GST-BLM, Eluted), and proteins resistant to elution were released by boiling in SDS-PAGE sample buffer (GST, Boiled; GST-BLM, Boiled). Input, eluted, and released proteins were analyzed for BLM, hRAD51, PARP, and Ku70 by Western blotting. (e) hRAD51 coimmunoprecipitates with BLM from nuclear lysates. Nuclear lysates from SOAS-2 cells (Input) were precleared and immunoprecipitated with nonspecific (IgG) or anti-hRAD51 (α-RAD51) antibody, and the immunoprecipitates were analyzed for BLM and hRAD51 by SDS-PAGE and Western blotting, as described in Materials and Methods.
Mentions: To determine how BLM associates with the PML NB, we prepared nuclear matrices from proliferating WI-38 cells using either of two methods: (a) standard high-salt extraction of nuclei, or (b) extraction at lower ionic strength followed by amine modification, which avoids the potential artifact of nonspecific salt precipitation (Wan et al. 1999). Proteins in the matrix and other fractions were analyzed by Western blotting. Regardless of the preparation method, BLM was found predominantly in the nuclear matrix fraction, cofractionating with the nuclear matrix marker protein lamin B (Spector 1993; Lamond and Earnshaw 1998; Wan et al. 1999; Fig. 3, a and b). BLM also fractionated with the nuclear matrix in VA-13 and HT1080 tumor cells (not shown).

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