Article |
Address correspondence to Judith Campisi, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Mailstop 84-171, Berkeley, CA 94720. Tel.: (510) 486-4416. Fax: (510) 486-4545. email: jcampisi{at}lbl.gov
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Abstract |
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Key Words: DNA damage; DNA repair; DNA replication; H2AX; S phase checkpoint
Abbreviations used in this paper: BS, Bloom syndrome; BSF, Bloom syndrome fibroblast; HM, helicase mutant; HU, hydroxyurea; NHF, normal human fibroblast; PML, promyelocytic leukemia protein.
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Introduction |
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Many genes that are mutated in human disorders are structurally or functionally conserved in lower eukaryotes, including yeast (Frei and Gasser, 2000b). Nonetheless, human disease genes rarely correct phenotypes resulting from mutations in the corresponding yeast gene. A notable exception is BLM and WRN, which complement defects in SGS1, the single RECQ homologue in the budding yeast Saccharomyces cerevisiae. BLM or WRN suppresses the hyper-recombination phenotype of sgs1 mutant yeast, although only BLM restores the slow growth and hydroxyurea (HU) resistance of top1:sgs1 double mutants (Yamagata et al., 1998). The HU sensitivity of sgs1 mutants is noteworthy because HU inhibits ribonucleotide reductase, thereby hindering DNA replication fork progression (Koc et al., 2003). Like BS cells, sgs1 mutants are genomically unstable, accumulating chromosome translocations, chromosome deletions, and sister chromatid exchanges, the most common chromosomal abnormality in BS (Onoda et al., 2000). Fission yeast (Schizosaccharomyces pombe) show similar phenotypes upon inactivation of its single RECQ homologue, RQH1/RAD12. Rqh1/rad12 mutants are hypersensitive to HU (Stewart et al., 1997), showing chromosomal abnormalities and hyper-recombination after exposure. Thus, yeast BLM orthologues may be cell cycledependent sensors and/or early responders to DNA damage. Indeed, recent findings suggest that Sgs1p signals to the S phase checkpoint machinery, intriguingly through proteinprotein interactions outside the helicase domain (Frei and Gasser, 2000b).
Indirect evidence suggests that BLM may likewise play a role in sensing and repairing DNA damage in mammalian cells (van Brabant et al., 2000); for example, the recent observation that Epstein Barr virustransformed BS lymphoblasts fail to form NBS1/MRE11 foci after exposure to HU (Franchitto and Pichierri, 2002). Here, we examine the role of BLM in the DNA damage response using human fibroblasts with intact damage checkpoints. We show that BS cells, or normal cells that express a helicase-defective BLM, die by apoptosis in response to diverse genotoxins. This sensitivity was limited to S phase, during which time damage rapidly mobilized BLM from its location in promyelocytic leukemia protein (PML) bodies to DNA breaks. We show that BLM is required for rapid recruitment of BRCA1 and NBS1 to the damage, but interestingly, this recruitment did not require BLM helicase activity. Of particular importance, BS cells deficient in p53 function did not die when damaged in S phase, nor did they delay BRCA1/NBS1 recruitment into foci. We suggest that our findings help explain the growth retardation and cancer predisposition of BS.
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Results |
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We next used retroviruses to introduce a control (insertless) vector or one that expresses either wild-type BLM or a mutant protein (helicase mutant [HM]) harboring a single amino acid substitution (K695T) that abolishes helicase activity (Neff et al., 1999). Wild-type or HM BLM is toxic when overexpressed in untransformed cells (Yankiwski et al., 2000; unpublished data). We therefore selected an uncloned population of low expressers from mass infected cultures (see Materials and methods). Western analysis confirmed that BLM is undetectable in control BS cells and that the retrovirus expressed wild-type and mutant BLM at levels similar to that of endogenous BLM in normal cells (Fig. 1 a). Immunostaining showed that >90% of BS cells expressed the retroviral proteins, and that they localized primarily to PML nuclear bodies (Fig. 1 b), the sites to which endogenous BLM localizes when it is expressed in S and G2 (Bischof et al., 2001b).
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Of significance, both asynchronous (not depicted) and S phase (Fig. 3 c) BSFs underwent apoptosis when treated with HU, which stalls replication forks (Koc et al., 2003). At 1030 mM, a 1-h exposure to HU caused as much cell death in 6 h as a 1-h exposure to etoposide (Fig. 3, a and b). A lower dose (1 mM), which still retards replication forks, also caused apoptosis, albeit after a longer interval (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200304016/DC1), consistent with the report that BS lymphoblasts die 36 h after exposure to 2 mM HU (Franchitto and Pichierri, 2002). This result suggests that BSFs are hypersensitive to replication stress.
Slow growth of BLM-deficient cells
hTERT-immortalized BSFs proliferated slowly relative to hTERT-expressing NHFs (doubling time 80100 h versus 40 h; Fig. 4 a), as did unmodified BSFs (not depicted). BLM complementation accelerated the growth of BSFs, whereas the HM mutant retarded the growth of NHFs (Fig. 4 a). These growth rate differences affected the kinetics of S phase entry after synchronization. We followed S phase entry after release from a G1/S block using 1-h pulses with BrdU. Greater than 80% of NHF and BSF + BLM cells incorporated BrdU 1 h after release, but <50% of BSF and NHF + HM cells did so. However, 3 h after release, >80% of BSF and NHF + HM cells incorporated BrdU (Fig. 4, b and c). These findings indicate that the slow growth of BSF and NHF + HM cells is not due to a smaller growth fraction. Moreover, their slower entry into and exit from S phase (Fig. 4 b) suggest that the slow growth is not due solely to an increase in G1, consistent with the cell cycle distributions of asynchronous cells determined by flow cytometry (unpublished data). We therefore adjusted the synchronization protocol in subsequent experiments (see Materials and methods) and assessed sensitivity to etoposide and HU when >80% of cells were in early S (Fig. 4 c). Under these conditions, BSF and NHF + HM cells showed
10-fold more apoptosis than NHF or BSF + BLM cells (Fig. 4, d and e). Unmodified BSF cells also underwent extensive apoptosis when given HU during S phase (Fig. S2 a, available at http://www.jcb.org/cgi/content/full/jcb.200304016/DC1), indicating that the cell death we observed was not due to hTERT expression. We conclude that BLM is crucial for cell survival during replication stress.
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As expected (Paull et al., 2000; Bischof et al., 2001b; Furuta et al., 2003), undamaged NHFs in early S showed diffuse H2AX and punctate BLM immunostaining (Fig. 6 a). When these cells were given bleomycin, which causes random double strand breaks (Kaufmann and Kies, 1998), multiple
H2AX foci formed in most cells within 1 h (Fig. 6, ac). Many (
70%) of these foci costained for BLM (Fig. 6, a and b). When the cells were given HU, multiple
H2AX foci also formed, indicating that some stalled forks acquired double strand breaks (Ward and Chen, 2001; Xu and Stern, 2003). BLM localized to nearly 90% of these foci (Fig. 6, a and b). BLM localized to
H2AX foci rapidly, lagging behind
H2AX focus formation by only a few minutes (Fig. 6 c). Unmodified NHFs (lacking hTERT) (Fig. S2 b) and complemented BS cells (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200304016/DC1) also showed rapid
H2AX/BLM colocalization in response to HU. Because bleomycin breaks DNA randomly, whereas HU causes breaks at replication forks, these results suggest that BLM localizes to replication forks that develop double strand breaks. It is possible, however, that BLM localizes to any double strand break during S phase. Whatever the case, immunoprecipitation and Western analysis showed that
70% of
H2AX coprecipitated with BLM when Jurkat cells were given HU (Fig. 6 d), suggesting that BLM and
H2AX reside in a common complex after replication forks stall. Moreover, >70% of the BLM/
H2AX foci that formed after HU treatment did not contain PML (unpublished data), consistent with our finding that BLM dissociates from PML bodies in a fraction of asynchronous cells treated with agents that break DNA (Bischof et al., 2001b). Together, these findings suggest that BLM is recruited from PML bodies to associate with double strand breaks during S phase.
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BRCA1 was shown to reside in a complex with BLM in a fraction of damaged, asynchronous HeLa cells (Wang et al., 2000). Immunoprecipitation showed that NBS1 also resides in a complex with BLM in asynchronous Jurkat cells treated with bleomycin and NHFs treated with HU in S phase (Fig. 7, f and g). Taken together, these findings suggest that BLM acts upstream of NBS1 and BRCA1 to assemble repair complexes at damaged replication forks.
Despite failure to form BRCA1 or NBS1 foci in early S, BSFs formed these foci later in S phase (68 h after release from G1/S) (not depicted). Moreover, although many BSFs did not form BRCA1/NBS1 foci 1 h after replication forks stalled, they formed these foci 6 h later (Fig. 7, ce). Thus, BLM deficiency delayed, but did not abrogate, BRCA1 and NBS1 recruitment into foci. Of interest, NHFs expressing the HM mutant were completely normal in assembling BRCA1/NBS1 foci during early S and 1 h after replication forks stalled (Fig. 7 b). Thus, despite dominant-negative activity with respect to growth and apoptosis, the HM mutant did not have dominant-negative activity with respect to BRCA1/NBS1 focus formation. This finding suggests that BLM has a structural role, which does not depend on its helicase activity, in assembling BRCA1/NBS1 complexes.
p53-deficient BSFs rapidly assemble NBS1 and BRCA1 foci after replication stress
In response to DNA damage, p53 halts cell cycle progression, presumably to allow repair, or induces apoptosis or senescence if the damage is severe (Ryan et al., 2001). Recent findings suggest that p53 may directly modulate repair or interact with repair proteins (Lu et al., 2003; Maser and DePinho, 2003; Rubbi and Milner, 2003). Because p53 mediated the apoptotic response of BSFs to damaged replication forks (Fig. 5), we asked whether p53 prevented the rapid assembly of BRCA1/NBS1 foci in BSFs. In contrast to BSFs with wild-type p53, 7080% of BSFs with mutant p53 (p53-V143A) assembled BRCA1 and NBS1 foci 1 h after HU stalled replication forks (Fig. 8, a and b). Loss of p53 function also partially restored the ability of undamaged BSF to form NBS1, but not BRCA1, foci in S phase (Fig. 8 b). NHFs with mutant p53, in contrast, showed no change in their relatively low incidence of apoptosis (Fig. 5 a), or ability to form BRCA1/NBS1 foci (Fig. 8, c and d) 1 h after HU stalled replication forks. These findings suggest that p53 acts downstream of BLM, delaying BRCA1/NBS1 focus formation in the absence of BLM.
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Discussion |
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In response to damage during S phase, BLM promptly left the PML bodies and associated with DNA double strand breaks, as assessed by loss of costaining with PML and gain of costaining with H2AX. The kinetics of costaining with
H2AX, and ability of HU to cause BLM relocalization, suggests that BLM rapidly localizes to double strand breaks at replication forks. These results, and the finding that BSFs fail to rapidly assemble BRCA1 or NBS1 foci after damage, suggest that BLM may be an important initial coordinator of processes that ensure the faithful repair of damage during DNA replication (Fig. 9). In the absence of BLM function, the majority of cells damaged during S phase die by apoptosis. We hypothesize that the survivors may repair the damage by suboptimal, low fidelity processes and thus may harbor a high incidence of the chromosomal abnormalities characteristic of BS cells.
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BS cells are prone to apoptosis
Stalled forks can develop double strand breaks, which typically cause senescence in NHFs (Robles and Adami, 1998). However, we observed no evidence of senescence when BSFs experienced damage during replication; rather, the cells died by apoptosis. Thus, BLM deficiency renders cells highly susceptible to apoptosis, suggesting a possible explanation for the pre- and postnatal growth retardation seen in BS. Endogenous oxidative metabolism continually generates DNA lesions (Ames, 1998), which can stall replication forks and generate double strand breaks. Under these conditions, BLM and other proteins must act quickly to repair the damage. In the absence of BLM, many cells may fail to repair the damage rapidly enough, whereupon p53 signals those cells to die. Individuals with BS may continually lose cells, owing to excessive apoptosis, particularly during pre- and postnatal development, when cell proliferation is extensive. Excessive apoptosis would leave many tissues with chronic cellular insufficiency, and hence small size, thereby explaining the pre- and postnatal growth retardation. BS is rare among humans, and it is hypothesized that many BS embryos die in utero (German, 1997). Consistent with this idea, most blm-/- mice die before birth due to massive apoptosis (Chester et al., 1998).
Role of BLM in BRCA1 and NBS1 focus formation
BLM expands the list of proteins that associate with H2AX at DNA breaks (Tauchi et al., 2002; Shang et al., 2003; Xu and Stern, 2003). BLM localized to
H2AX foci within minutes of their formation. As
H2AX interacts directly with NBS1 (Kobayashi et al., 2002), BLM may facilitate the NBS1
H2AX interaction. Consistent with this view, the HM mutant was proficient at recruiting BRCA1 and NBS1, despite its inability to prevent apoptosis after DNA damage during S phase. Thus, although BLM helicase activity is essential for preventing cell death, it is dispensable for rapid BRCA1/NBS1 recruitment. These findings suggest that BLM has both an enzymatic role and a structural role in the rapid response to damage during S phase. Interestingly, WRN may also have both catalytic and structural roles in DNA repair (Chen et al., 2003).
BSFs that survive damage during S phase eventually formed BRCA1 and NBS1 foci, albeit with delayed kinetics. Thus, BLM is not unconditionally necessary for BRCA1 and NBS1 focus formation, but rather is necessary only for the rapid response. In the absence of BLM, another RECQ helicase may substitute for BLM, but its recruitment may be less efficient or require events that take several hours to complete. BLM also enhanced the ability of BRCA1 and NBS1 to form foci during S phase in undamaged cells. However, of the BRCA1 and NBS1 foci that formed during S phase, only 2030% colocalized with BLM in PML bodies. Why then are BRCA1 and NBS1 foci so rare in BS cells? One possibility is that BRCA1 and NBS1 transiently shuttle into the PML body, where BLM facilitates modifications that allow these proteins to form foci outside the PML body. Consistent with this possibility, BLM may be necessary for NBS1 phosphorylation (Franchitto and Pichierri, 2002).
Yeast RECQ homologues have been proposed to act as sensors of damage encountered during DNA synthesis (Frei and Gasser, 2000a,b), a role also ascribed to mammalian BRCA1 and the MRE11/RAD50/NBS1 complex. Loss of any of these proteins sensitizes cells to DNA damage and gives rise to a mutator phenotype. BLM and MRE11/RAD50/NBS1 were recently identified as part of a large complex, which BRCA1 was proposed to anchor to the nuclear matrix (Wang et al., 2000). Our previous finding that BLM associates tightly with the nuclear matrix (Bischof et al., 2001b), and our finding that BLM is required for rapid recruitment of BRCA1 to damage-induced foci, raises the possibility that BLM provides the scaffold for assembly of the BRCA1 repair complex. Assembly of this complex may prevent the death of cells with damaged replication forks. This possibility is consistent with the finding that BLM is rapidly cleaved during apoptosis induced by severe genotoxic stress (Bischof et al., 2001a).
Role of p53 in BS phenotypes
p53 was crucial for the apoptotic death of BS cells. This apoptosis was not accompanied by an increase in BAX or p21 expression (not depicted), genes for which p53 is a known transactivator. Thus, p53 may induce apoptosis independent of its transactivation activity, consistent with the finding that p53 is transcriptionally inactive during S phase (Gottifredi et al., 2001; Takimoto and El-Deiry, 2001). p53 may mediate the death of damaged BSFs by directly inducing mitochondria-mediated apoptosis (Mihara et al., 2003), or by virtue of it transrepression activity (Ryan et al., 2001).
Loss of p53 function allowed BS cells to survive replication stress, but the survivors may acquire chromosomal aberrations (unpublished data). This scenario provides a plausible explanation for the extraordinarily high cancer incidence seen in BS. The excessive apoptosis that likely occurs in the proliferative tissues of BS individuals would also create a chronic need for cell replacement and hence cell division, which increases the risk of oncogenic mutations. Because mutations in the p53 pathway prevent the death of BS cells, there is intense selection for BS cells that lose p53 function. Loss of p53 function alone is a strong risk factor for genomic instability and the development of cancer (Donehower, 1996). However, BLM deficiency may exacerbate the loss of genomic integrity due to faulty repair by BLM-deficient BRCA1/NBS1 complexes. In BS cells with wild-type p53 function, these complexes assemble after a delay, perhaps owing to time required to recruit a substitute helicase. In cells with defective p53 function, however, these complexes assemble rapidly, but may lack the substitute helicase and repair may be faulty. This model (Fig. 9) is consistent with recent findings showing that BLM and p53 deficiencies synergize to up-regulate homologous recombination (Sengupta et al., 2003).
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Materials and methods |
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Retroviruses
We produced amphotropic retroviruses and infected cells as previously described (Kim et al., 1999; Itahana et al., 2002). We cloned the wild-type BLM cDNA into pMSCVhyg (CLONTECH Laboratories, Inc.) and HM mutant cDNA (Neff et al., 1999) into pBabe-puro (Morgenstern and Land, 1990).
BLM complemented BSFs and HM-expressing NHFs
hTERT-expressing cells were infected on three consecutive days with control (insertless), BLM-expressing, or BLM-HMexpressing viruses. To select for low expressers, we cultured cells in antibiotics (0.75 µg/ml puromycin or 100 µg/ml hygromycin) for 12 d, allowed recovery for 1421 d, and then selected again for 7 d.
Synchronization
For G1 synchronization, we seeded cells in serum-containing medium and, 24 h later, shifted them to medium containing 0.2% serum for 7296 h. We then added medium containing 10% serum for 46 h. To synchronize cells at the G1/S boundary and accommodate the different doubling times of NHFs and BSFs, we serum deprived (0.2%) the cells for 7296 h, cultured for 8 (NHF and BSF + BLM) or 12 h (BSF and NHF + HM) in 10% serum, and then cultured in medium containing 10% serum and either 2 µg/ml aphidicolin or 2 mM HU for 12 (NHF and BSF + BLM) or 16 h (BSF and NHF + HM). To release from the G1/S block, cells were washed extensively and cultured in drug-free, serum-containing medium for 1 (NHF and BSF + BLM) or 3 h (BSF and NHF + HM) before addition of genotoxic agents. To synchronize cells with mimosine, we cultured cells in low serum (0.2%) for 72 h, added 10% serum for 6 h, and then added 0.25 mM mimosine for 16 h. The cells were washed extensively and then cultured in drug-free serum containing medium for 3 h before addition of genotoxic agents.
Antibodies
We used affinity-purified rabbit (Neff et al., 1999) or goat anti-BLM (C-18 and K-20; Santa Cruz Biotechnology, Inc.), rabbit (Neomarkers) or mouse (ab-1; Oncogene Research Products) anti-BRCA1, rabbit anti-NBS1 (Novus Biologicals; Oncogene Research Products), mouse (PG-M3; Santa Cruz Biotechnology, Inc.) or rabbit (Chemicon) anti-PML, mouse anti-actin (Chemicon), mouse anti-p53, and mouse anti-tubulin (ab-1; Oncogene Research Products) antibodies. Rabbit anti-H2AX was previously described (Burma et al., 2001). We detected BrdU incorporation by immunofluorescence, using a commercially available kit (Roche).
Immunofluorescence
We seeded 2 x 104 cells in four-well chamber slides, fixed with ice-cold methanol for 20 min at -20°C or 4% paraformaldehyde for 5 min at room temperature, permeabilized with 0.5% Triton X-100 for 5 min, and blocked with 10% serum from the secondary antibody species. We incubated cells in primary antibodies for 1 h at room temperature or overnight at 4°C and in fluorochrome-conjugated secondary antibodies for 0.51 h. To visualize DNA, the last wash contained 0.4 µg/ml DAPI. We mounted slides in Vectashield and viewed by epifluorescence. Images were captured with a CCD camera and merged using Adobe Photoshop®.
Drug treatments and apoptosis assay
We treated cells with etoposide, bleomycin, or HU (Sigma-Aldrich) for 1 h at the indicated concentrations, washed, and cultured in drug-free medium for 6 h. We detected apoptosis by collapse of mitochondrial membrane potential using the MitoCapture reagent (Biovision) (Kaminker et al., 2001), morphology of nuclei after DAPI staining, and caspase-3 activity using a commercially available kit (Oncogene Research Products). In brief, cell lysates were incubated with a synthetic caspase-3 substrate conjugated to a colorimetric tag in the presence or absence of the caspase-3 inhibitor DEVD-CHO for 5 h. Caspase-3 activity is proportional to optical density at 405 nm. The graph shows activity in arbitrary units.
Western analysis
We prepared protein lysates in 5% SDS, separated 30 µg protein using 415% SDS-PAGE, transferred the proteins to a nylon membrane, blocked, and incubated with primary and secondary antibodies, as previously described (Bischof et al., 2001b). We detected secondary antibodies by ECL (Amersham Biosciences).
Immunoprecipitation
We employed a protocol previously described (Yannone et al., 2001). We precleared 5001,000 µg cell lysate with ultra-link beads (Pierce Chemical Co.), incubated the supernatant with either goat anti-IgG or anti-BLM antibodies overnight at 4°C, and clarified the supernatants by centrifugation. We incubated the supernatants with fresh ultra-link beads, washed the beads, and released the bound proteins using Laemmli sample buffer. We separated the released proteins by 415% SDS-PAGE, as previously described (Bischof et al., 2001b).
Online supplemental material
The supplemental material (Figs. S1S5) is available at http://www.jcb.org/cgi/content/full/jcb.200304016/DC1. The supplemental figures show that low dose HU causes apoptosis in BSFs (Fig. S1), unmodified BSFs behave similarly to hTERT-immortalized BSFs (Fig. S2), BLM localizes to g-H2AX foci in BLM-complemented cells (Fig. S3), BLM localizes to g-H2AX foci after replication stress in BRCA1 mutant or NBS1-deficient cells (Fig. S4), and BRCA1 and NBS1 foci form in BS cells after low dose HU or mimosine synchronization (Fig. S5).
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Acknowledgments |
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This work was supported by National Institutes of Health (NIH) research grant AG11658 (J. Campisi), NIH training grant AG00226, and Californian Breast Cancer Research Program award 8FB-0148 (A.R. Davalos).
Submitted: 3 April 2003
Accepted: 31 July 2003
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