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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, PML, hRAD51, and RP-A localization in cells in G0 or late S/G2. WI-38 cells were made quiescent (Quiescent), or released from a G1/S arrest for 8 h (Late S/G2). Cells were stained 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) hRAD51 and PML localization in quiescent cells. (e–h) hRAD51 and BLM localization in cells in late S/G2. (i–l) RP-A and BLM localization in cells in late S/G2. Bars, ∼10 μm.
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Figure 2: BLM, PML, hRAD51, and RP-A localization in cells in G0 or late S/G2. WI-38 cells were made quiescent (Quiescent), or released from a G1/S arrest for 8 h (Late S/G2). Cells were stained 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) hRAD51 and PML localization in quiescent cells. (e–h) hRAD51 and BLM localization in cells in late S/G2. (i–l) RP-A and BLM localization in cells in late S/G2. Bars, ∼10 μm.

Mentions: In quiescent cells, hRAD51 immunostaining was largely diffuse throughout the nucleus, but ∼30% of nuclei contained hRAD51 foci (Fig. 2 a), 70–80% of which localized to PML NBs (Fig. 2, a–d). As cells approached late S/G2, hRAD51 staining intensity increased, and ∼40% of nuclei had >10 distinct RAD51/PML foci. More than half the RAD51 foci also contained BLM (Fig. 2, e–h), and, in about a third of the cells, 80–90% of the BLM foci contained hRAD51. Western blotting showed that hRAD51 was detectable in quiescent cells, but expression increased about fivefold as cells approached late S/G2 (not shown). Thus, as cells progressed through late S and G2, an increasing fraction assembled nuclear foci that contained PML, BLM, and hRAD51. These results are summarized in Table . Because 40% of cells in late S/G2 had RAD51 foci, >50% of which contained BLM, and 80–90% of BLM foci in late S/G2 localized to PML NBs, we deduce that roughly 15–20% of late S/G2 nuclei contained all three proteins (PML, RAD51, RAD51).


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, PML, hRAD51, and RP-A localization in cells in G0 or late S/G2. WI-38 cells were made quiescent (Quiescent), or released from a G1/S arrest for 8 h (Late S/G2). Cells were stained 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) hRAD51 and PML localization in quiescent cells. (e–h) hRAD51 and BLM localization in cells in late S/G2. (i–l) RP-A and BLM localization in cells in late S/G2. Bars, ∼10 μm.
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Related In: Results  -  Collection

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Figure 2: BLM, PML, hRAD51, and RP-A localization in cells in G0 or late S/G2. WI-38 cells were made quiescent (Quiescent), or released from a G1/S arrest for 8 h (Late S/G2). Cells were stained 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) hRAD51 and PML localization in quiescent cells. (e–h) hRAD51 and BLM localization in cells in late S/G2. (i–l) RP-A and BLM localization in cells in late S/G2. Bars, ∼10 μm.
Mentions: In quiescent cells, hRAD51 immunostaining was largely diffuse throughout the nucleus, but ∼30% of nuclei contained hRAD51 foci (Fig. 2 a), 70–80% of which localized to PML NBs (Fig. 2, a–d). As cells approached late S/G2, hRAD51 staining intensity increased, and ∼40% of nuclei had >10 distinct RAD51/PML foci. More than half the RAD51 foci also contained BLM (Fig. 2, e–h), and, in about a third of the cells, 80–90% of the BLM foci contained hRAD51. Western blotting showed that hRAD51 was detectable in quiescent cells, but expression increased about fivefold as cells approached late S/G2 (not shown). Thus, as cells progressed through late S and G2, an increasing fraction assembled nuclear foci that contained PML, BLM, and hRAD51. These results are summarized in Table . Because 40% of cells in late S/G2 had RAD51 foci, >50% of which contained BLM, and 80–90% of BLM foci in late S/G2 localized to PML NBs, we deduce that roughly 15–20% of late S/G2 nuclei contained all three proteins (PML, RAD51, RAD51).

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