BLM and the FANC proteins collaborate in a common pathway in response to stalled replication forks - PubMed (original) (raw)

BLM and the FANC proteins collaborate in a common pathway in response to stalled replication forks

Pietro Pichierri et al. EMBO J. 2004.

Abstract

Fanconi anaemia (FA) and Bloom syndrome (BS) are autosomal recessive diseases characterised by chromosome fragility and cancer proneness. Here, we report that BLM and the FA pathway are activated in response to both crosslinked DNA and replication fork stall. We provide evidence that BLM and FANCD2 colocalise and co-immunoprecipitate following treatment with either DNA crosslinkers or agents inducing replication arrest. We also find that the FA core complex is necessary for BLM phosphorylation and assembly in nuclear foci in response to crosslinked DNA. Moreover, we show that knock-down of the MRE11 complex, whose function is also under the control of the FA core complex, enhances cellular and chromosomal sensitivity to DNA interstrand crosslinks in BS cells. These findings suggest the existence of a functional link between BLM and the FA pathway and that BLM and the MRE11 complex are in two separated branches of a pathway resulting in S-phase checkpoint activation, chromosome integrity and cell survival in response to crosslinked DNA.

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Figures

Figure 1

Figure 1

Loss of BLM or FA pathway results in enhanced cell death following crosslinked DNA and replication arrest. (A) MMC sensitivity of the indicated genotypes. (B) Photoactivated 8-MOP sensitivity of the indicated genotypes. (C, D) HU sensitivity of the indicated genotypes (see Materials and methods). The lymphoblasts used were GM3657 (WT), HSC536 (FA-C), HSC536 Corr (FA-C+FANCC), HSC72 (FA-A), EUFA143L (FA-G), GM16752 (FA-D2), GM3403 (BS1), GM3403_BLM_ (BS1+BLM) and ZG (BS). The SV40-transformed fibroblasts used were MRC5 (WT), PD20 (FA-D2), PD20 315 (FA-D2+FANCD2), PD332 (FA-C), PD352 (FA-G), PD352+FANCG (FA-G+FANCG), GM8505 (BS) and GM8505+BLM (BS+BLM). Data are the mean±s.d. from three independent experiments. Data were analysed by χ2 test; the differences in sensitivity between wild-type and FA or BS cells were statistically significant (P<0.01, χ2 test).

Figure 2

Figure 2

The BLM protein and FANCD2 respond similarly to DNA crosslink induction and replication fork arrest. (A) Representative images taken after 6 h of recovery from MRC5 fibroblasts. (B) ICL- and replication arrest-dependent assembly of BLM and FANCD2 nuclear foci. Wild-type fibroblasts (MRC5 and BJ) and lymphoblasts (GM3657) were exposed to photoactivated 8-MOP (ICL), HU or UVC light and analysed at the indicated time points. (C) BLM phosphorylation in response to crosslinked DNA and replication arrest. Upper blot: MRC5 fibroblasts were lysed 4 h after treatment with 10 μM photoactivated 8-MOP (ICL), 2 mM HU or 40 J/m2 UVC, and BLM phosphorylation was analysed by the shift in electrophoretic mobility as seen on 5% Tris-glycine gel. Lower blot: BLM phosphorylation was visualised by 32Pi metabolic labelling followed by autoradiography. The BLM band shift is not visible on the lower blot because of the higher percentage of acrylamide used for the SDS–PAGE. (D) FANCD2 monoubiquitination and phosphorylation in response to stalled replication fork. Cellular extracts prepared 4 h after exposure of wild-type cells (MRC5) to photoactivated 8-MOP (ICL), HU or UVC were analysed for the presence of the large isoform of FANCD2 (FANCD2-L) and for FANCD2 phosphorylation by Western blot analysis. Phosphorylation was assessed by observing the shift in the electrophoretic mobility of the FANCD2 doublet and confirmed by reversion of the band shift following PPase treatment.

Figure 3

Figure 3

BLM and FANCD2 colocalise in response to crosslinked DNA and replication fork arrest. (A) ICL- and replication fork arrest-dependent BLM/FANCD2 colocalisation in wild-type cells. MRC5 cells (wild type) were exposed to photoactivated 8-MOP or UVC and immunofluorescence was carried out 6 h later. In the lower row is presented a nucleus in which BLM and FANCD2 foci are both present but not colocalising. (B) Co-IP of BLM and FANCD2. Cellular extracts prepared 6 h after exposure of wild-type or FA-D2 (PD20) cells to photoactivated 8-MOP (ICL), HU or UVC were immunoprecipitated and immunoprecipitates were analysed by Western blot. IgG: Extract prepared from untreated MRC5 cells immunoprecipitated with normal rabbit IgG. Lower panel: The supernatant from the first immunoprecipitation was used to re-immunoprecipitate FANCD2 and evaluate the fraction not bound to BLM. (C) Co-IP of BLM with the activated (i.e. ubiquitinated) form of FANCD2. MRC5 (wild type) and FA-G cells were transiently transfected with an empty vector or with a plasmid expressing HA-tagged ubiquitin and 36 h later exposed to photoactivated 8-MOP (ICL) or HU. Cellular extracts prepared 6 h after treatment were immunoprecipitated with a polyclonal antibody against BLM and immunoprecipitates were analysed by Western blot using anti-BLM, anti-FANCD2 and anti-HA-tag antibodies. IgG: Extract prepared from untreated MRC5 cells immunoprecipitated with normal rabbit IgG. Note that the FANCD2 doublet as well as the BLM band shift is not visible on the blots because of the percentage of acrylamide used for the SDS–PAGE (7% instead of 5%).

Figure 4

Figure 4

Integrity of the FA core complex is required for ICL-dependent BLM phosphorylation. (A) BLM relocalisation in FA cells following ICL and replication arrest. Wild-type (FA-G+FANCG), FA-G and FA-D2 SV40-immortalised fibroblasts were exposed to photoactivated 8-MOP (ICL) or to HU and immunofluorescence was carried out 6 h later. (B) Analysis of the BLM relocalisation in wild-type (WT), FA and phenotypically complemented cells. The analysis was carried out at 6 h post-treatment and similar results were obtained at 4 and 8 h. Data are the mean±s.d. from three independent experiments. *Statistically significant (P<0.01, χ2 test). (C) BLM phosphorylation assessed by Western blot following treatment with photoactivated 8-MOP (ICL), HU or UVC in wild-type and FA lymphoblasts. (D) Evaluation of BLM phosphorylation in FA-C cells. Cells were exposed to HU or photoactivated 8-MOP (ICL) and analysed for the presence of BLM phosphorylation by Western blot. (E) Analysis of BLM phosphorylation at different times after induction of ICL by 8-MOP. (F) Cells were exposed to UVC or photoactivated 8-MOP, and FANCD2 phosphorylation was visualised by 32Pi metabolic labelling followed by autoradiography. The FANCD2 band shift is not visible on the lower blot because of the higher percentage of acrylamide used in the SDS–PAGE (7% instead of 5%). Unless specified, BLM phosphorylation was evaluated as band shift 4 h after the indicated treatment.

Figure 5

Figure 5

The FA core complex and FANCD2 migrate to chromatin in response to replication arrest in a BLM-independent manner. (A) FANCD2 relocalisation in BS cells following UVC or HU. Wild-type (WT) and BS hTert-immortalised fibroblasts were exposed to photoactivated 8-MOP (ICL) or to HU and immunofluorescence was carried out 6 h later. (B) Analysis of the FANCD2 relocalisation in wild-type (WT) and BS hTert-immortalised fibroblasts and EBV-transformed lymphoblasts. The analysis was carried out at 6 h post-treatment but consistent results were also obtained at other sampling times. Data are the mean±s.d. from three independent experiments. (C) FANCD2 monoubiquitination in BS lymphoblasts. Cellular extracts were prepared 4 h after exposure of cells to photoactivated 8-MOP (ICL) or UVC. A sample irradiated with 10 Gy of IR (γ) served as positive control. (D) FANCD2 phosphorylation analysed by 32Pi metabolic labelling. A cellular extract prepared from cells irradiated with 10 Gy of γ-rays was included as a positive control for FANCD2 phosphorylation. Note that the FANCD2 doublet is not visible on the blots because of the percentage of acrylamide used for SDS–PAGE (7.5% instead of 5%). (E) Cellular extracts prepared 6 h after exposure of BS and BLM-complemented lymphoblasts to photoactivated 8-MOP (ICL) or UVC were biochemically fractionated and the free nucleoplasmic (N) and the insoluble chromatin (C) fractions were sequentially analysed for the presence of FANC proteins. Similar results were obtained analysing FANCG distribution (data not shown). Immunoblotting using anti-topoisomerase II (TopoII) and anti-CHK2 antibodies served as control for the fractionation procedure and as loading control.

Figure 6

Figure 6

BLM and MRE11 contribute through two separate pathways to ensure cell viability and chromosome integrity following induction of crosslinked DNA. (A, B) ICL sensitivity of the indicated genotypes. Data are the mean±s.d. from three independent experiments. Results were found significant by the χ2 test (P<0.01). (C) Quantification of the photoactivated 8-MOP-induced chromosome breakage. Analysis was carried out as described in Materials and methods after exposure to 500 nM 8-MOP followed by 10 kJ/m2 UVA. Data are the mean±s.d. from three independent experiments. Results were significant as judged by the Student's _t_-test (P<0.01). (D) Analysis of the S-phase checkpoint activation following ICL induction. Replicative DNA synthesis was assessed at different time points after ICL induction with photoactivated 8-MOP. (E) Sensitivity to HU-induced replication arrest of the indicated genotypes. Data are the mean±s.d. from three independent experiments. Results were found significant by the χ2 test (P<0.01). In the case of siRNA-treated cells, exposure to genotoxins was always performed 72 h after transfection.

Figure 7

Figure 7

Model showing the cooperation of the BLM and the FA pathways in the response to replication arrest induced by either DNA crosslinks or UVC and HU exposure. In response to HU or UVC (A), stall of the replication machinery triggers activation of both the BLM–MRE11 and the FA branches. Possibly, the BLM–MRE11 branch is required for direct resolution of the replication arrest, whereas the FA pathway might be required for the subsequent recombinational-based replication rescue. FANCD2–BLM interaction might be functional at this step. In response to ICL-dependent replication fork arrest (B), the upstream FA core complex is activated to trigger the FA-MRE11 and the BLM branches of the pathway. Both the branches might be involved in the correct execution of the recombination-based rescue of replication, ensuring cell viability and chromosomal integrity.

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