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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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Cell cycle–dependent localization of BLM. Cells were synchronized, immunostained for BLM or PML, stained for nuclear DNA (DAPI), and pulsed (1 h) with [3H]thymidine to determine the percentage of cells in S phase (% LN), as described in Materials and Methods. (a and b) BLM antibody specificity. Proliferating BS fibroblasts (HG2654, shown; GM11492F, not shown) were stained with DAPI (a) to visualize nuclei and the anti-BLM antibody (b). (c) BLM foci during the cell cycle. WI-38 cells were arrested in G0 (Q) and then stimulated with serum for 8 h (Q,8) to enrich for cells in mid-G1. Alternatively, cells were arrested at the G1/S boundary (HU) and released for varying intervals to enrich for cells in mid-S (HU,4), late S/G2 (HU,8), G2/M/early G1 (HU,10), or G1/early S (HU,12). The percent of LN was determined in parallel cultures. Nuclei (≥200 per data point) were scored for the presence of >10 BLM foci. (d–k) BLM and PML were identified by immunostaining using fluorescein isothiocyanate (green) or Texas red secondary antibodies. Red and green fluorescent images were superimposed (MERGE). Nuclei were identified by DAPI staining. (d) PML localization in quiescent cells. (e) BLM localization in quiescent cells. (f) Merged image of PML and BLM localization in quiescent cells. (g) DAPI staining of nuclei in d–f. (h) PML localization in cells in late S/G2. (i) BLM localization in cells in late S/G2. (j) Merged image of PML and BLM localization in cells in late S/G2. (k) DAPI staining of nuclei in h–k. Bars, ∼10 μm.
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Figure 1: Cell cycle–dependent localization of BLM. Cells were synchronized, immunostained for BLM or PML, stained for nuclear DNA (DAPI), and pulsed (1 h) with [3H]thymidine to determine the percentage of cells in S phase (% LN), as described in Materials and Methods. (a and b) BLM antibody specificity. Proliferating BS fibroblasts (HG2654, shown; GM11492F, not shown) were stained with DAPI (a) to visualize nuclei and the anti-BLM antibody (b). (c) BLM foci during the cell cycle. WI-38 cells were arrested in G0 (Q) and then stimulated with serum for 8 h (Q,8) to enrich for cells in mid-G1. Alternatively, cells were arrested at the G1/S boundary (HU) and released for varying intervals to enrich for cells in mid-S (HU,4), late S/G2 (HU,8), G2/M/early G1 (HU,10), or G1/early S (HU,12). The percent of LN was determined in parallel cultures. Nuclei (≥200 per data point) were scored for the presence of >10 BLM foci. (d–k) BLM and PML were identified by immunostaining using fluorescein isothiocyanate (green) or Texas red secondary antibodies. Red and green fluorescent images were superimposed (MERGE). Nuclei were identified by DAPI staining. (d) PML localization in quiescent cells. (e) BLM localization in quiescent cells. (f) Merged image of PML and BLM localization in quiescent cells. (g) DAPI staining of nuclei in d–f. (h) PML localization in cells in late S/G2. (i) BLM localization in cells in late S/G2. (j) Merged image of PML and BLM localization in cells in late S/G2. (k) DAPI staining of nuclei in h–k. Bars, ∼10 μm.

Mentions: The BLM antibody has been characterized (Ishov et al. 1999; Neff et al. 1999; Zhong et al. 1999). As expected, it failed to stain BS fibroblasts (Fig. 1, a and b), but identified 10–30 nuclear foci in WI-38 fibroblasts (Fig. 1, d–k), confirming its specificity. To semiquantitatively assess BLM foci, we scored the fraction of WI-38 cells with >10 discernible foci per nucleus (Fig. 1 c). Quiescent cells had faint diffuse nuclear staining (not shown), but ∼10% had >10 faint foci (Fig. 1 c). This staining pattern persisted as cells progressed through G1 (Q,8; Fig. 1 c). However, as cells progressed through S (after release from HU), the number (Fig. 1 c) and intensity (not shown) of BLM foci rose, increasing until most cells were in late S or G2 (HU,8; Fig. 1 c). At this time, half the cells had >10 (generally 20–40) bright BLM foci, which declined as cells entered the next cell cycle (HU,10; Fig. 1 c). The intensity and number of BLM foci were always heterogeneous, possibly due to the unavoidable loss of tight synchrony or the dynamic nature of the foci. We did not detect BLM in nucleoli, as reported for some cells (Yankiwski et al. 2000).


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)

Cell cycle–dependent localization of BLM. Cells were synchronized, immunostained for BLM or PML, stained for nuclear DNA (DAPI), and pulsed (1 h) with [3H]thymidine to determine the percentage of cells in S phase (% LN), as described in Materials and Methods. (a and b) BLM antibody specificity. Proliferating BS fibroblasts (HG2654, shown; GM11492F, not shown) were stained with DAPI (a) to visualize nuclei and the anti-BLM antibody (b). (c) BLM foci during the cell cycle. WI-38 cells were arrested in G0 (Q) and then stimulated with serum for 8 h (Q,8) to enrich for cells in mid-G1. Alternatively, cells were arrested at the G1/S boundary (HU) and released for varying intervals to enrich for cells in mid-S (HU,4), late S/G2 (HU,8), G2/M/early G1 (HU,10), or G1/early S (HU,12). The percent of LN was determined in parallel cultures. Nuclei (≥200 per data point) were scored for the presence of >10 BLM foci. (d–k) BLM and PML were identified by immunostaining using fluorescein isothiocyanate (green) or Texas red secondary antibodies. Red and green fluorescent images were superimposed (MERGE). Nuclei were identified by DAPI staining. (d) PML localization in quiescent cells. (e) BLM localization in quiescent cells. (f) Merged image of PML and BLM localization in quiescent cells. (g) DAPI staining of nuclei in d–f. (h) PML localization in cells in late S/G2. (i) BLM localization in cells in late S/G2. (j) Merged image of PML and BLM localization in cells in late S/G2. (k) DAPI staining of nuclei in h–k. Bars, ∼10 μm.
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Related In: Results  -  Collection

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Figure 1: Cell cycle–dependent localization of BLM. Cells were synchronized, immunostained for BLM or PML, stained for nuclear DNA (DAPI), and pulsed (1 h) with [3H]thymidine to determine the percentage of cells in S phase (% LN), as described in Materials and Methods. (a and b) BLM antibody specificity. Proliferating BS fibroblasts (HG2654, shown; GM11492F, not shown) were stained with DAPI (a) to visualize nuclei and the anti-BLM antibody (b). (c) BLM foci during the cell cycle. WI-38 cells were arrested in G0 (Q) and then stimulated with serum for 8 h (Q,8) to enrich for cells in mid-G1. Alternatively, cells were arrested at the G1/S boundary (HU) and released for varying intervals to enrich for cells in mid-S (HU,4), late S/G2 (HU,8), G2/M/early G1 (HU,10), or G1/early S (HU,12). The percent of LN was determined in parallel cultures. Nuclei (≥200 per data point) were scored for the presence of >10 BLM foci. (d–k) BLM and PML were identified by immunostaining using fluorescein isothiocyanate (green) or Texas red secondary antibodies. Red and green fluorescent images were superimposed (MERGE). Nuclei were identified by DAPI staining. (d) PML localization in quiescent cells. (e) BLM localization in quiescent cells. (f) Merged image of PML and BLM localization in quiescent cells. (g) DAPI staining of nuclei in d–f. (h) PML localization in cells in late S/G2. (i) BLM localization in cells in late S/G2. (j) Merged image of PML and BLM localization in cells in late S/G2. (k) DAPI staining of nuclei in h–k. Bars, ∼10 μm.
Mentions: The BLM antibody has been characterized (Ishov et al. 1999; Neff et al. 1999; Zhong et al. 1999). As expected, it failed to stain BS fibroblasts (Fig. 1, a and b), but identified 10–30 nuclear foci in WI-38 fibroblasts (Fig. 1, d–k), confirming its specificity. To semiquantitatively assess BLM foci, we scored the fraction of WI-38 cells with >10 discernible foci per nucleus (Fig. 1 c). Quiescent cells had faint diffuse nuclear staining (not shown), but ∼10% had >10 faint foci (Fig. 1 c). This staining pattern persisted as cells progressed through G1 (Q,8; Fig. 1 c). However, as cells progressed through S (after release from HU), the number (Fig. 1 c) and intensity (not shown) of BLM foci rose, increasing until most cells were in late S or G2 (HU,8; Fig. 1 c). At this time, half the cells had >10 (generally 20–40) bright BLM foci, which declined as cells entered the next cell cycle (HU,10; Fig. 1 c). The intensity and number of BLM foci were always heterogeneous, possibly due to the unavoidable loss of tight synchrony or the dynamic nature of the foci. We did not detect BLM in nucleoli, as reported for some cells (Yankiwski et al. 2000).

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