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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 focus formation after DNA damage. Proliferating WI-38 (a–c) and AT-2SF (c) cells were X-irradiated (5 Gy) or UV-irradiated (1.6 J/m2/s) and immunostained for BLM at the indicated intervals thereafter. BLM foci were counted in 200 nuclei per point. (a) BLM foci increase after IR. Nuclei were scored for the presence of >10 or <10 BLM foci. (b) Effect of IR versus UV. Cells were unirradiated (−IR) or irradiated with X-rays (+IR) or UV (+UV). 10 h later, nuclei were scored for the presence of 0–10, 11–20, or >20 BLM foci. (c) BLM foci formation in irradiated AT cells. Proliferating AT-2SF or WI-38 cells were X-irradiated. 10 h later, nuclei were scored for the presence of 11–20 or >20 BLM foci.
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Figure 6: BLM focus formation after DNA damage. Proliferating WI-38 (a–c) and AT-2SF (c) cells were X-irradiated (5 Gy) or UV-irradiated (1.6 J/m2/s) and immunostained for BLM at the indicated intervals thereafter. BLM foci were counted in 200 nuclei per point. (a) BLM foci increase after IR. Nuclei were scored for the presence of >10 or <10 BLM foci. (b) Effect of IR versus UV. Cells were unirradiated (−IR) or irradiated with X-rays (+IR) or UV (+UV). 10 h later, nuclei were scored for the presence of 0–10, 11–20, or >20 BLM foci. (c) BLM foci formation in irradiated AT cells. Proliferating AT-2SF or WI-38 cells were X-irradiated. 10 h later, nuclei were scored for the presence of 11–20 or >20 BLM foci.

Mentions: BLM foci were heterogeneously distributed in asynchronous cultures, with few nuclei containing >10 foci (Fig. 6 a). However, after X-irradiation (5 Gy) nuclei with >10 BLM foci rose, whereas those with <10 foci declined, in a time- (Fig. 6 a) and dose- (not shown) dependent manner. 10 h after IR, nuclei with >10 BLM foci were four- to fivefold more prevalent than in control cultures. Moreover, at this time 15–20% of irradiated nuclei had >20 BLM foci, whereas such nuclei were rare in controls (Fig. 6 b). The IR-induced peak in BLM foci (Fig. 6 a) coincided with the IR-induced peak in BLM protein and G2 delay (Fig. 4). Etoposide similarly increased BLM foci coincident with BLM protein and a G2 delay (not shown). By contrast, BLM foci did not rise after UV irradiation (Fig. 6 b), which did not increase BLM expression (Fig. 5 e) and delays cells in G1 (Kaufmann and Kies 1998). Compared with controls, UV-irradiated cultures had more nuclei with ≤10 BLM foci, and fewer with 11–20 BLM foci (Fig. 6 b), consistent with their more prominent G1 delay. Thus, BLM foci increased in response to agents that cause DSBs, similar to IRIF, and the increase coincided with the G2 delay.


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 focus formation after DNA damage. Proliferating WI-38 (a–c) and AT-2SF (c) cells were X-irradiated (5 Gy) or UV-irradiated (1.6 J/m2/s) and immunostained for BLM at the indicated intervals thereafter. BLM foci were counted in 200 nuclei per point. (a) BLM foci increase after IR. Nuclei were scored for the presence of >10 or <10 BLM foci. (b) Effect of IR versus UV. Cells were unirradiated (−IR) or irradiated with X-rays (+IR) or UV (+UV). 10 h later, nuclei were scored for the presence of 0–10, 11–20, or >20 BLM foci. (c) BLM foci formation in irradiated AT cells. Proliferating AT-2SF or WI-38 cells were X-irradiated. 10 h later, nuclei were scored for the presence of 11–20 or >20 BLM foci.
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Related In: Results  -  Collection

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Figure 6: BLM focus formation after DNA damage. Proliferating WI-38 (a–c) and AT-2SF (c) cells were X-irradiated (5 Gy) or UV-irradiated (1.6 J/m2/s) and immunostained for BLM at the indicated intervals thereafter. BLM foci were counted in 200 nuclei per point. (a) BLM foci increase after IR. Nuclei were scored for the presence of >10 or <10 BLM foci. (b) Effect of IR versus UV. Cells were unirradiated (−IR) or irradiated with X-rays (+IR) or UV (+UV). 10 h later, nuclei were scored for the presence of 0–10, 11–20, or >20 BLM foci. (c) BLM foci formation in irradiated AT cells. Proliferating AT-2SF or WI-38 cells were X-irradiated. 10 h later, nuclei were scored for the presence of 11–20 or >20 BLM foci.
Mentions: BLM foci were heterogeneously distributed in asynchronous cultures, with few nuclei containing >10 foci (Fig. 6 a). However, after X-irradiation (5 Gy) nuclei with >10 BLM foci rose, whereas those with <10 foci declined, in a time- (Fig. 6 a) and dose- (not shown) dependent manner. 10 h after IR, nuclei with >10 BLM foci were four- to fivefold more prevalent than in control cultures. Moreover, at this time 15–20% of irradiated nuclei had >20 BLM foci, whereas such nuclei were rare in controls (Fig. 6 b). The IR-induced peak in BLM foci (Fig. 6 a) coincided with the IR-induced peak in BLM protein and G2 delay (Fig. 4). Etoposide similarly increased BLM foci coincident with BLM protein and a G2 delay (not shown). By contrast, BLM foci did not rise after UV irradiation (Fig. 6 b), which did not increase BLM expression (Fig. 5 e) and delays cells in G1 (Kaufmann and Kies 1998). Compared with controls, UV-irradiated cultures had more nuclei with ≤10 BLM foci, and fewer with 11–20 BLM foci (Fig. 6 b), consistent with their more prominent G1 delay. Thus, BLM foci increased in response to agents that cause DSBs, similar to IRIF, and the increase coincided with the G2 delay.

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