Phosphorylation of BLM, dissociation from topoisomerase IIIalpha, and colocalization with gamma-H2AX after topoisomerase I-induced replication damage

Abstract

Topoisomerase I-associated DNA single-strand breaks selectively trapped by camptothecins are lethal after being converted to double-strand breaks by replication fork collisions. BLM (Bloom's syndrome protein), a RecQ DNA helicase, and topoisomerase IIIalpha (Top3alpha) appear essential for the resolution of stalled replication forks (Holliday junctions). We investigated the involvement of BLM in the signaling response to Top1-mediated replication DNA damage. In BLM-complemented cells, BLM colocalized with promyelocytic leukemia protein (PML) nuclear bodies and Top3alpha. Fibroblasts without BLM showed an increased sensitivity to camptothecin, enhanced formation of Top1-DNA complexes, and delayed histone H2AX phosphorylation (gamma-H2AX). Camptothecin also induced nuclear relocalization of BLM, Top3alpha, and PML protein and replication-dependent phosphorylation of BLM on threonine 99 (T99p-BLM). T99p-BLM was also observed following replication stress induced by hydroxyurea. Ataxia telangiectasia mutated (ATM) protein and AT- and Rad9-related protein kinases, but not DNA-dependent protein kinase, appeared to play a redundant role in phosphorylating BLM. Following camptothecin treatment, T99p-BLM colocalized with gamma-H2AX but not with Top3alpha or PML. Thus, BLM appears to dissociate from Top3alpha and PML following its phosphorylation and facilitates H2AX phosphorylation in response to replication double-strand breaks induced by Top1. A defect in gamma-H2AX signaling in response to unrepaired replication-mediated double-strand breaks might, at least in part, explain the camptothecin-sensitivity of BLM-deficient cells.

FIG. 1.

Enhanced sensitivity and Top1-DNA complex formation in BLM-deficient fibroblasts exposed to camptothecin (CPT). (A) Cell survival following camptothecin exposure for 1 h was measured in BLM-deficient (PSNG13) and -complemented (PSNF5) cell lines by colony formation assay. (Inset) BLM and Top3α protein expression in PSNG13 and PSNF5 cells. (B) Exponentially growing PSNG13 or PSNF5 cells were exposed to 1 μM camptothecin for 1 h. Cesium chloride fractions from homogenized sarkosyl lysates were immunoblotted for Top1. (C) DNA-containing fractions were combined, serially diluted (2-, 5-, or 10-fold), and immunoblotted for Top1. (D) Quantitation of the serial dilutions of DNA-containing fractions were compared between PSNG13 and PSNF5 following camptothecin treatment (n = 3). Bars, standard deviations. (E) Top1 protein expression in PSNG13 and PSNF5 cellular lysates following a 1-h exposure to 1 μM camptothecin. (F) Proliferative status of control PSNF5 and PSNG13 cells as measured by BrdU staining and fluorescence-activated cell sorting analysis. Scattergrams shown are representative of at least four independent experiments.

FIG. 2.

Delayed appearance of H2AX phosphorylation (γ-H2AX) following replication-mediated DNA damage in BLM-deficient fibroblasts. (A) BLM-deficient (PSNG13) and -complemented (PSNF5) cells were exposed to 1 μM camptothecin for the indicated periods. Cells were then permeabilized and fixed using paraformaldehyde and ethanol. Fixed cells were stained with a γ-H2AX antibody and secondary antibody conjugated with a green fluorescent dye (Alexa 488). Reversal of γ-H2AX was examined following a 12-h treatment with 1 μM camptothecin. Nuclear outlines were traced from a parallel image with propidium iodide staining and superimposed on the γ-H2AX images. (B) Kinetics of appearance of γ-H2AX foci was quantitated in PSNG13 and PSNF5 cells after exposure to 1 μM camptothecin, 1 mM hydroxyurea (HU), or 1 G ionizing radiation. (C) Levels of γ-H2AX were assayed in BLM-deficient and -complemented cells exposed to 1 μM camptothecin for indicated times by Western blot analysis. Images are representative of at least three independent experiments. (D) Generation of γ-H2AX foci in primary healthy human fibroblasts (GM00037) and in primary Bloom syndrome patient fibroblasts (GM01492) following 1 μM camptothecin treatment for indicated times. (E) Quantitation of γ-H2AX foci in GM00037 and GM01492 cells treated with camptothecin. At least 30 nuclei from five separate fields were included for quantitation. CPT, camptothecin.

FIG. 3.

Changes in nuclear localization patterns of BLM, PML, and Top3α in BLM-complemented and -deficient fibroblasts. PSNG13 and PSNF5 cells were exposed to 1 μM camptothecin for 1 h or untreated (control). PSNF5 cells were stained with both Top3α (green) and BLM (red) antibodies (A) or with both PML (green) and BLM (red) antibodies (B) or with both PML (green) and Top3α (red) antibodies (C). (D) PSNG13 cells were probed with PML (red) and Top3α (green) antibodies. Note: The majority of PSNG13 cells failed to show punctate Top3α foci. The image presented for PSNG13 cells is representative of a small fraction of nuclei that showed clear Top3α and PML foci. Images displayed from a single nuclear structure are representative of at least three independent experiments. Fig. 7 and Table 1 show the quantitation of foci in response to camptothecin. CPT, camptothecin.

FIG. 4.

Phosphorylation of BLM on T99 in camptothecin-treated cells. (A) A peptide consisting of the phosphorylated T99 BLM (T99p-BLM) was used to generate antisera in rabbits. Crude serum was double-affinity purified and used in control experiments with extracts from PSNF5 and PSNG13 cells. Preimmunization sera and phospho-peptide column eluate were used as negative controls for PSNF5 lysates from cells treated with 1 mM hydroxyurea for 16 h. Extracts from PSNG13 cells treated with hydroxyurea were used as a negative control for the affinity-purified T99p-BLM antibody. (B) Total protein lysates from PSNF5 cells exposed to 1 μM camptothecin for 1 h or 6 h were probed by Western blotting with the T99p-BLM antibody. The 6-h lysate was also incubated with λ-protein phosphatase (λ-Pase) at 30°C for 60 min with or without protein phosphatase inhibitor (λ-Pase Inh, combination of sodium orthovanadate and sodium fluoride). The lower panel shows lysates probed with BLM antibody. (C) Immunofluoresence images of PSNG13 or PSNF5 cells stained with T99p-BLM antibody after hydroxyurea treatment for 16 h. Nuclear outlines were traced from a parallel image with propidium iodide staining and superimposed on the T99p-BLM images. (D) PSNF5 cells were exposed to 1 μM camptothecin as indicated. (E) Quantitation of T99p-BLM foci per nucleus from experiments using increasing concentrations of either camptothecin or hydroxyurea for indicated times in PSNF5 cells. At least 30 nuclei from three independent experiments were used in the analysis. HU, hydroxyurea; camptothecin, CPT.

FIG. 5.

Replication-dependent phosphorylation of BLM on T99. (A) Schematic representation of replication double-strand breaks (DSB) induced by camptothecin. The left side of the panel shows reversible Top1 cleavage complex trapped by camptothecin (black rectangle). The right side of the panel shows the Top1 cleavage complex on the replicating leading strand leads to a double-strand break by replication runoff (48, 62). Aphidicolin prevents the formation of replication double-strand breaks. (B) BLM-complemented PSNF5 cells were treated with 1 μM camptothecin for 1 h with or without pretreatment with 10 μM aphidicolin for 15 min. Cells were stained with the T99p-BLM antisera (green) and total BLM antibody (red). Nuclear images are representative of at least three independent experiments. (C) Graphical representation of the average number of T99p-BLM or BLM foci per nucleus after camptothecin or aphidicolin and camptothecin treatment in PSNF5 cells. At least 30 nuclei from three independent experiments were used in the analysis. CPT, camptothecin; APH, aphidicolin.

FIG. 6.

Phosphorylation of BLM on T99 in the absence of ATM, ATR, or DNA-PK in response to replication double-strand breaks. Fibroblasts from a healthy (GM00637) and an AT patient (GM05849) (A) or ATRkd and ATR wild-type fibroblasts (B) were compared by confocal microscopic analysis for the kinetics of appearance of T99p-BLM foci by camptothecin. ATRkd cells were pretreated with 1 μg/ml doxycycline (Doxy +) for 24 h to induce the expression of ATR kinase inactive, resulting in ATR kinase dominant-negative status. (C) The results of a quantitative focus analysis for at least 30 cells from two independent experiments are shown. (D) DNA-PK catalytic subunit (DNA-PKcs)-deficient (MO59J/Fus1) and DNA-PKcs-complemented (MO59J/Fus1) cell lines were also studied for the appearance of T99p-BLM foci.

FIG. 7.

Colocalization of BLM phosphorylated on T99 with γ-H2AX and dissociation of T99p-BLM from Top3α in response to replication double-strand breaks. (A) BLM-complemented PSNF5 cells were costained using T99-phospho-BLM (green) and γ-Η2AX (red) antibodies following a 1-h exposure to 1 μM camptothecin. (B) Costaining with T99-pBLM (green) and PML (red) antibodies. (C) Costaining with T99p-BLM (green) and Top3α (red). (D) Costaining with total BLM (red) and γ-H2AX (green). (E) Costaining with γ-H2AX (green) and PML (red). (F) Costaining with Top3α (green) and γ-H2AX (red). Confocal microscopy images are representative of at least three independent experiments. (G) Graphical representation of the percentage fraction of localization of either BLM or T99p-BLM with indicated proteins after camptothecin treatment. At least 30 nuclei were used for the analysis from three independent experiments. CPT, camptothecin.

FIG. 8.

Proposed dual role of BLM in DNA repair and signal transduction in response to replication-mediated DNA damage. Molecular Interaction Map (MIM) annotation conventions can be found at http://discover.nci.nih.gov/mim (3). (A) In unstressed cells, BLM is bound to Top3α in the PML nuclear bodies (1) (bindings between two molecular species are shown as double-headed arrows. The node on the line corresponds to the bimolecular complex (BLM-Top3α [1]). (B) Camptothecin binds to the Top1 cleavage complexes (Top1 CC) (the green node [2] is the CPT-Top1 CC complex) (47). CPT-trapped Top1 CC induces replication blocks (Repl. Block) (62) (3) (activations are shown as green line with open arrowhead). Replication blocks produce DNA damage (4) (conversions are shown as black arrow) (Repl. Damage is replication-mediated double-strand breaks). BLM-Top3α complexes bind to the blocked replication forks (red node) and prevent replication damage (5) by resolving (repairing) the replication blocks. (C) Replication damage induces the phosphorylation of histone H2AX on Ser 139 (6) and phosphorylation of BLM on Thr 99 (7) (phosphorylations are shown as blue arrows). BLM activates H2AX phosphorylation (8). We propose that phosphorylation of BLM on T99 (red node on the BLM phosphorylation line) inhibits its binding to Top3α (9). CPT, camptothecin.