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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 responds to IR. Proliferating WI-38 cells were X-irradiated (IR) with 5 Gy (a, b, and d) or 0–10 Gy (c). RNA and protein were isolated, or cells were harvested for flow cytometry, at the indicated intervals (h) thereafter. BLM mRNA was measured by quantitative PCR using QM as a control; BLM protein was assessed by Western blotting using α-tubulin (Tubulin) as a control. A value of one was assigned to the normalized levels of BLM mRNA and protein in unirradiated cells (0 h). Autoradiograms of the Western analyses are shown above the histograms. (a) BLM mRNA after IR. (b) BLM protein after IR. (c) IR dose response. Cells were analyzed for BLM protein 4 h (autoradiogram) or 8 h (autoradiogram, histogram) after irradiation. (d) Cell cycle arrest after IR. Cells were analyzed for DNA content by flow cytometry. The G1 (2N) and G2 (4N) peaks are indicated and the fraction of cells in G1, S, and G2/M is given in the text.
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Figure 4: BLM responds to IR. Proliferating WI-38 cells were X-irradiated (IR) with 5 Gy (a, b, and d) or 0–10 Gy (c). RNA and protein were isolated, or cells were harvested for flow cytometry, at the indicated intervals (h) thereafter. BLM mRNA was measured by quantitative PCR using QM as a control; BLM protein was assessed by Western blotting using α-tubulin (Tubulin) as a control. A value of one was assigned to the normalized levels of BLM mRNA and protein in unirradiated cells (0 h). Autoradiograms of the Western analyses are shown above the histograms. (a) BLM mRNA after IR. (b) BLM protein after IR. (c) IR dose response. Cells were analyzed for BLM protein 4 h (autoradiogram) or 8 h (autoradiogram, histogram) after irradiation. (d) Cell cycle arrest after IR. Cells were analyzed for DNA content by flow cytometry. The G1 (2N) and G2 (4N) peaks are indicated and the fraction of cells in G1, S, and G2/M is given in the text.

Mentions: BLM mRNA was quantified using real-time RT-PCR (Heid et al. 1996) and QM as a constitutively expressed control mRNA (Dimri et al. 1996). IR induced a modest, transient rise in BLM mRNA, amounting to a fourfold increase within 2 h, before returning to the unirradiated (control) level (Fig. 4 a). BLM protein also increased 2–4 h after IR, but in contrast to the mRNA, continued to accumulate for 8–10 h, peaking at 10-fold over the control level. Peak BLM levels persisted for 4–6 h (12–14 h after IR; Fig. 4 b) before declining to the control level (24 h after IR; not shown). A lower dose (1 Gy) of IR induced less BLM (threefold over control), but a higher dose (10 Gy) did not increase BLM further (Fig. 4 c). Peak BLM induction by IR coincided with the arrest of cell proliferation, detected by flow cytometry. Unirradiated cultures maintained a cell cycle distribution typical of asynchronous populations (55% G1, 36% S, 9% G2/M; Fig. 4 d). Irradiated cultures, by contrast, accumulated cells in G1 (53%) and G2/M (42%) within 12 h, at which time fewer than 5% of cells were in S phase (Fig. 4 d). Because the cells have a finite replicative life span, even the early passage cultures used here contain 15–20% senescent cells, which have a G1 DNA content (Campisi 1997). Thus, the fraction of irradiated cells that transiently arrested in G1 was likely <40%, whereas the fraction that arrested in G2 was likely >50%. Cells resumed proliferation 24 h after IR (not shown). These results raise the possibility that BLM is induced by DNA damage. Alternatively, because BLM is expressed predominantly in late S/G2, its accumulation after IR may reflect the accumulation of cells in G2.


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 responds to IR. Proliferating WI-38 cells were X-irradiated (IR) with 5 Gy (a, b, and d) or 0–10 Gy (c). RNA and protein were isolated, or cells were harvested for flow cytometry, at the indicated intervals (h) thereafter. BLM mRNA was measured by quantitative PCR using QM as a control; BLM protein was assessed by Western blotting using α-tubulin (Tubulin) as a control. A value of one was assigned to the normalized levels of BLM mRNA and protein in unirradiated cells (0 h). Autoradiograms of the Western analyses are shown above the histograms. (a) BLM mRNA after IR. (b) BLM protein after IR. (c) IR dose response. Cells were analyzed for BLM protein 4 h (autoradiogram) or 8 h (autoradiogram, histogram) after irradiation. (d) Cell cycle arrest after IR. Cells were analyzed for DNA content by flow cytometry. The G1 (2N) and G2 (4N) peaks are indicated and the fraction of cells in G1, S, and G2/M is given in the text.
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Figure 4: BLM responds to IR. Proliferating WI-38 cells were X-irradiated (IR) with 5 Gy (a, b, and d) or 0–10 Gy (c). RNA and protein were isolated, or cells were harvested for flow cytometry, at the indicated intervals (h) thereafter. BLM mRNA was measured by quantitative PCR using QM as a control; BLM protein was assessed by Western blotting using α-tubulin (Tubulin) as a control. A value of one was assigned to the normalized levels of BLM mRNA and protein in unirradiated cells (0 h). Autoradiograms of the Western analyses are shown above the histograms. (a) BLM mRNA after IR. (b) BLM protein after IR. (c) IR dose response. Cells were analyzed for BLM protein 4 h (autoradiogram) or 8 h (autoradiogram, histogram) after irradiation. (d) Cell cycle arrest after IR. Cells were analyzed for DNA content by flow cytometry. The G1 (2N) and G2 (4N) peaks are indicated and the fraction of cells in G1, S, and G2/M is given in the text.
Mentions: BLM mRNA was quantified using real-time RT-PCR (Heid et al. 1996) and QM as a constitutively expressed control mRNA (Dimri et al. 1996). IR induced a modest, transient rise in BLM mRNA, amounting to a fourfold increase within 2 h, before returning to the unirradiated (control) level (Fig. 4 a). BLM protein also increased 2–4 h after IR, but in contrast to the mRNA, continued to accumulate for 8–10 h, peaking at 10-fold over the control level. Peak BLM levels persisted for 4–6 h (12–14 h after IR; Fig. 4 b) before declining to the control level (24 h after IR; not shown). A lower dose (1 Gy) of IR induced less BLM (threefold over control), but a higher dose (10 Gy) did not increase BLM further (Fig. 4 c). Peak BLM induction by IR coincided with the arrest of cell proliferation, detected by flow cytometry. Unirradiated cultures maintained a cell cycle distribution typical of asynchronous populations (55% G1, 36% S, 9% G2/M; Fig. 4 d). Irradiated cultures, by contrast, accumulated cells in G1 (53%) and G2/M (42%) within 12 h, at which time fewer than 5% of cells were in S phase (Fig. 4 d). Because the cells have a finite replicative life span, even the early passage cultures used here contain 15–20% senescent cells, which have a G1 DNA content (Campisi 1997). Thus, the fraction of irradiated cells that transiently arrested in G1 was likely <40%, whereas the fraction that arrested in G2 was likely >50%. Cells resumed proliferation 24 h after IR (not shown). These results raise the possibility that BLM is induced by DNA damage. Alternatively, because BLM is expressed predominantly in late S/G2, its accumulation after IR may reflect the accumulation of cells in G2.

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