From the Life Sciences Division, Lawrence Berkeley
National Laboratory, Berkeley California 94720 and the
¶ Department of Molecular Medicine, The Rayne Institute, Guy's,
Kuig's and St. Thomas' School of Medicine, King's
College, London SE5 9NU, United Kingdom
Received for publication, July 20, 2000, and in revised form, December 13, 2000
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ABSTRACT |
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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
that is presumed to function in mammalian DNA replication,
recombination, or repair. We show here that BLM, but not the related
RECQ-like helicase WRN, is rapidly cleaved in cells undergoing
apoptosis. BLM was cleaved to 47- and 110-kDa major fragments, with
kinetics similar to the apoptotic cleavage of poly(A)DP-ribose
polymerase. BLM cleavage was prevented by a caspase 3 inhibitor and did
not occur in caspase 3-deficient cells. Moreover, recombinant BLM was
cleaved to 47- and 110-kDa fragments by caspase 3, but not caspase 6, in vitro. The caspase 3 recognition sequence
412TEVD415 was verified by mutating
aspartate 415 to glycine and showing that this mutation rendered BLM
resistant to caspase 3 cleavage. Cleavage did not abolish the BLM
helicase activity but abolished BLM nuclear foci and the association of
BLM with condensed DNA and the insoluble matrix. The results suggest
that BLM, but not WRN, is an early selected target during the execution
of apoptosis.
Apoptosis, or programmed cell death, is critical for many
biological processes, including tissue morphogenesis and homeostasis, the development of immunity and host defense mechanisms, and
elimination of damaged or potentially neoplastic cells (1-3).
Moreover, defective apoptosis may cause or exacerbate a variety of
human diseases, including Alzheimer's and Huntington's diseases,
autoimmunity, and cancer (4-6). Cells that are dying by apoptosis
share many cytological and molecular features, regardless of the
initiating signal. Cytological changes include cytoplasmic and nuclear
shrinkage, plasma membrane blebbing, and chromatin condensation. At the
molecular level, a family of cysteine proteases, termed caspases,
selectively cleaves a series of protein substrates. Initiator caspases,
such as caspases 8 and 9, initiate a proteolytic cascade that
eventually culminates in the cleavage and activation of downstream
caspases, such as caspases 3 and 6. The downstream or execution
caspases then cleave selected target proteins (7-9). Subsequently, a
caspase-activated deoxyribonuclease
(CAD1) cleaves genomic DNA
(10), at which point cell death is imminent.
Most caspases recognize a four-amino acid sequence having a C-terminal
aspartate, after which they cleave. Caspases are highly selective in
their target proteins and generally cleave at only one or a few
specific sites within the target. Caspase-mediated cleavage can lead to
either functional activation or inactivation of the target protein,
which presumably facilitates the orderly execution of apoptosis
(7-13). The most prominent execution caspase is caspase 3, also known
as CPP32, Yama, and apopain. Several proteins that sense DNA damage
and/or participate in DNA repair have been identified as caspase 3 substrates. These proteins include ATM (14), RAD51 (15, 16), the
catalytic subunit of DNA-dependent protein kinase
(DNA-PKcs) (17), and poly(A)DP-ribose polymerase (PARP) (18, 19).
Caspase 3 cleavage effectively inactivates these proteins and,
subsequently, CAD degrades DNA (10). Thus, in advance of DNA
fragmentation, caspases ensure that cells destined for apoptotic death
inactivate proteins involved in maintaining genomic integrity, thereby
assuring the orderly execution of apoptosis.
Recently, a family of genes related to Escherichia coli RECQ
has been implicated in maintaining genomic integrity in human cells
(20). RECQ encodes a DNA helicase that participates in homologous recombination and suppresses illegitimate recombination (21,
22). At least five human RECQ-like genes have been
identified: RECQL (23, 24), BLM (25),
WRN (26), RECQL4, and RECQL5 (27).
Among these, BLM was the first to be linked to a hereditary disease (25). Defects in BLM cause Bloom's syndrome (BS),
an autosomal recessive disorder characterized by multiple
abnormalities, including immunodeficiency, pre- and post-natal growth
retardation, and a high incidence of cancer (28). Cancer is the primary
cause of death in BS and generally occurs before the third decade of life. Cells from individuals with BS are hypermutable. BS cells accumulate chromatid gaps and breaks and, most prominently, numerous sister chromatid exchanges (28, 29).
BLM encodes a 1417-amino acid 3' Because of its importance in maintaining genomic stability, we asked
whether BLM was among the proteins targeted for selective degradation
during the execution phase of apoptosis. We show here that BLM, but not
the related RECQ-like protein WRN, is proteolytically cleaved at a
single site in cells induced to undergo apoptosis by multiple stimuli,
and identify caspase 3 as the responsible protease. BLM cleavage was an
early apoptotic event that did not abrogate BLM helicase activity but
caused disappearance of BLM foci and detachment from condensed DNA and
the insoluble matrix. Thus, BLM, but not WRN, is targeted for
degradation during the early execution phase of apoptosis.
Reagents--
Tosyl-L-lysine chloromethyl ketone
(TLCK) and leupeptin were purchased from Roche Diagnostics,
acetyl-Tyr-Val-Ala-Asp fluoromethyl ketone (ZVAD-FMK) was from Enzyme
System Products, and acetyl-Asp-Glu-Val-Asp aldehyde (Ac-DEVD-CHO) was
from BD PharMingen. Other reagents were molecular biology or cell
culture grade, purchased as indicated, and prepared according to the
manufacturer's instructions.
Antibodies--
The affinity-purified rabbit anti-N-terminal BLM
(33), rabbit anti-C-terminal BLM (35), and rabbit anti-WRN (36)
antibodies have been described previously. Anti-tubulin (Ab 1) was from
Oncogene Science, anti-PARP (H-250) was from Santa Cruz Biotechnology, anti-Ku70 (clone N3H10) was from NeoMarkers, and anti-Fas (clone CH-11)
was from MBL International Corp. Fluorescence- or horseradish peroxidase-conjugated secondary antibodies were from Vector
Laboratories or Bio-Rad.
Cell Culture and Induction of Apoptosis or Necrosis--
Cells
were obtained from the American Type Culture Collection and cultured
under standard conditions in RPMI 1640 or Dulbecco's modified Eagle's
medium (Life Technologies, Inc.) and 10% fetal calf serum. To induce
apoptosis, Jurkat cells (1 × 106 cells/ml) were
incubated with 100 ng/ml anti-FAS antibody (MBL International Corp.) or
2 µM staurosporine (Sigma Chemical Co.), HeLa cells were
incubated with 30 ng/ml tumor necrosis factor- Nuclear Extracts, Total Cell Lysates, and Western
Analyses--
Nuclear extracts were prepared from 5-10 × 106 cells by rapid salt extraction. Briefly, cells were
washed twice in ice-cold phosphate buffered saline (PBS) and stored at
Digitonin Extraction--
Digitonin extraction was performed as
described by Adam et al. (40). Briefly, 3-6 h after
induction of apoptosis, cells were washed twice in ice-cold PBS,
pelleted at 190 × g for 5 min, and gently resuspended
in PBS containing 1% digitonin (Sigma). After a 5-min incubation on
ice, cells were pelleted to separate cytosolic proteins (supernatant)
from insoluble proteins (pellet). Detergent was removed from
supernatant and pellet proteins by precipitating in methanol-chloroform
(41). The precipitate was solubilized in 2× SDS-PAGE sample buffer,
and the proteins were analyzed by Western blotting.
Immunofluorescence--
Jurkat cells (1 × 105)
were collected using a cytospin, fixed, and stained at room temperature
as described (42). Cells were incubated with primary antibodies for
2 h and secondary antibodies for 1 h. Slides were mounted in
Vectashield containing DAPI (4',6-diamidino-2-phenyl-indole, Vector
Laboratories) and viewed by epifluorescence. The images were captured
using a cooled charge-coupled device camera and merged using Canvas (Deneba).
In Vitro Cleavage of BLM--
Glutathione
S-transferase (GST) and GST-BLM fusion protein were
expressed in Sf9 insect cells using recombinant baculoviruses and purified using a commercial kit (Life Technologies, Inc.). Nuclear
lysates were prepared as described (36, 43), clarified by
centrifugation, and incubated for 1 h at 4 °C with
glutathione-Sepharose 6-CL B resin (Amersham Pharmacia Biotech). The
slurry was transferred to a column, and washed with 50 column volumes
of PBS plus 0.2% Nonidet P-40 and 50 volumes of PBS. Proteins were
eluted with 20 mM glutathione, 100 mM Tris-HCl,
pH 8.0, 150 mM NaCl, 1 mM dithiothreitol, 10%
glycerol (elution buffer), and migrated as single bands on
silver-stained SDS-PAGE. Protein concentrations were determined by
Bio-Rad assay. Recombinant activated caspases were expressed in
E. coli and prepared as described (44). Purified GST-BLM (20 nM) and caspase (1 µM) were incubated for
1 h at 37 °C in elution buffer. Reactions were terminated by
adding an equal volume of 2× SDS-PAGE sample buffer or directly
assayed for helicase activity.
Helicase Assay--
Helicase assays were performed as described
by Huang et al. (45). Briefly, activity was detected by
displacement of a 32P-5'-labeled 20-bp oligonucleotide from
a partial 20-bp/46-bp DNA duplex in which the 46-bp oligonucleotide was
unlabeled. The reaction (20 µl) was incubated for 10 min at 37 °C
and terminated by rapid cooling and addition of 5× loading buffer (2%
SDS, 50 mM EDTA, 3% bromphenol blue, 3% xylene cyanol,
40% glycerol). The displaced oligonucleotide was separated from the
partial duplex by 12% nondenaturing PAGE. Where indicated, proteins
and probe were denatured prior to assay by heating to 95 °C for 5 min.
Generation of Caspase-resistant Mutant and in Vitro
Translation--
Replacement of aspartate with glycine at position 415 was carried out by overlapping PCR. Briefly, pSG5-Myc-BLM1, containing the full-length BLM cDNA with an N-terminal Myc epitope tag was digested with EcoRI and BamHI. The 1.4-kb
fragment containing the site to be mutagenized was subcloned into
pBluescriptKS+ (Stratagene). Two oligonucleotides spanning
the site were synthesized (BLM-5', CTTCTAACGGAAGTAGGTTTTAATAAAAGTGATGCC
and BLM-3', GGCATCACTTTTATTAAAACCTACTTCCGTTAGAAG). Polymerase chain
reaction (PCR) was used to amplify a 1.3-kb fragment using the M13
forward and BLM-3' primer and a 200-bp fragment using the M13 Reverse
and BLM-5' primer. The fragments were mixed at equal molar
concentrations, and a 1.5-kb fragment was amplified using the M13
Forward and Reverse primers. The PCR product was digested with
EcoRI and BamHI, subcloned in pBluescriptKS, and sequenced using the T3 primer. The mutagenized 1.4-kb
EcoRI-BamHI fragment was cloned into
pSG5-Myc-BLM1 that was partially digested with EcoRI and
BamHI to obtain full-length D415A-BLM. In vitro translation was performed using the coupled TnT reticulocyte lysate system (Promega) and [35S]methionine (Redivue, Amersham
Pharmacia Biotech). The translation products were untreated or
incubated with 0.1 µM activated recombinant caspase for
1 h at 37 °C, solubilized in SDS-PAGE sample buffer, separated
by 4-15% SDS-PAGE, and visualized by autoradiography.
BLM Is Cleaved during Apoptosis--
To determine whether BLM is
cleaved during apoptosis, we initially used a well established model
system, human Jurkat leukemia T cells induced to undergo apoptosis by a
FAS-monoclonal antibody (46). Nuclear extracts were prepared from cells
treated with anti-FAS for varying intervals and analyzed by Western
blotting, using antibodies raised against either the N-terminal 431 amino acids (33) or the C-terminal 375 amino acids of BLM
(35).
Cleavage of BLM was first evident 2-4 h after addition of anti-FAS,
progressing to near completion over the next 8-10 h (Fig. 1A). The 159-kDa BLM protein
was cleaved to a 110- to 115-kDa fragment detected by the C-terminal
antibody (Fig. 1A), and a 45- to 50-kDa fragment, detected
by the N-terminal antibody (see Fig. 2).
Subsequent experiments (see Fig. 4) showed that these fragments were
112 and 47 kDa, respectively. In striking contrast to BLM, WRN, a
related RECQ-like helicase, was not cleaved during anti-FAS-mediated
apoptosis (Fig. 1B). Likewise, Ku70, a component of
DNA-dependent protein kinase, which is critical for
repairing DNA double-strand breaks by nonhomologous end-joining,
remained intact (Fig. 1C). Thus, cleavage of BLM was an
early and relatively selective event during apoptosis.
The initiation of BLM cleavage coincided with the initiation of PARP
cleavage (Fig. 1D), a well established early event in the
execution of apoptosis (18, 19). On the other hand, although PARP was
completely cleaved 6-9 h after addition of anti-FAS (Fig. 1D), BLM required about 12 h for complete cleavage
(Fig. 1A). The more rapid completion of PARP cleavage may
reflect differences in the enzymes or kinetics by which PARP and BLM
are cleaved or differences in their accessibility to apoptotic
proteases. Like the PARP apoptotic fragments, the BLM apoptotic
fragments were stable for several hours after the initiation of apoptosis.
BLM cleavage was not limited to anti-FAS-induced apoptosis, or to
Jurkat cells. HeLa cells undergo rapid apoptosis in response to tumor
necrosis factor-
In contrast to its fate during apoptosis, BLM was not cleaved when
cells were induced to undergo necrotic cell death. Jurkat cells were
treated with anti-FAS to induce apoptotic death, or oligomycin, which
causes death by necrosis (37). Oligomycin caused a rapid loss of
intracellular ATP, characteristic of necrotic death (37), compared with
the slower loss of ATP caused by anti-FAS, characteristic of apoptotic
death (Fig. 2C). Western analysis of extracts prepared
6 h later, when ATP levels were comparably low in the oligomycin
and anti-FAS-treated cells, showed that BLM cleavage occurred only
during anti-FAS-induced apoptosis; there was little or no BLM cleavage
during oligomycin-induced necrosis (Fig. 2D). Moreover,
oligomycin, which to a large extent inhibits apoptosis, also to a large
extent prevented BLM degradation (Fig. 2D).
Taken together, these results suggest that BLM is an early target for
selective apoptosis-induced proteolysis.
Identification of the Protease That Cleaves BLM--
To identify
the protease responsible for apoptotic cleavage of BLM, we treated
Jurkat cells with anti-FAS in the presence of protease inhibitors. BLM
cleavage was not prevented by inhibitors of serine proteases, leupeptin
(100 or 200 µM; Fig.
3A, lanes 5,
6) or tosyl-L-lysine chloromethyl ketone (TLCK)
(100 µM; Fig. 3A, lane 3). A high
concentration of TLCK (200 µM) inhibited apoptotic BLM
cleavage (Fig. 3A, lane 4). TLCK has been shown
to also inhibit the activity and activation of caspases in
vitro (47), and a high concentration of TLCK (200 µM) was shown to induce necrosis in Jurkat cells without
features of apoptosis (48). In contrast to leupeptin and TLCK, caspase
inhibitors (49) prevented apoptotic BLM cleavage at moderate to low
concentrations. This was true for the broad-range inhibitor ZVAD-FMK
(10 and 20 µM; Fig. 3A, lanes 7,
8) and the caspase 3/caspase 7 inhibitor Ac-DEVD-CHO (100 µM; Fig. 3A, lane 9). These results
suggest that the cleavage of BLM during apoptosis depends on the
activity of a caspase 3 or caspase 7-like enzyme. Caspases 3 and 7 have
identical recognition sequences. However, caspase 3 is the more likely
candidate for cleaving BLM in vivo, because it, in contrast
to caspase 7, is found in the nucleus (50) where BLM resides.
We determined that caspases 3 and 7 were capable of cleaving BLM by
incubating purified recombinant BLM and activated caspases in
vitro. Recombinant BLM was a fusion protein coupled to glutathione S-transferase (GST-BLM, ~188 kDa; see Fig.
4) and recombinant caspases were the
activated forms of the executioner caspases 3, 6, and 7. Caspases 3 and
7, but not caspase 6, cleaved GST-BLM (Fig. 3B). In both
cases, BLM was cleaved to a 76-kDa fragment, detected by the N-terminal
antibody (Fig. 3B) and silver staining (Fig. 3C),
and a 112-kDa fragment detected by silver staining (Fig.,
3C). The size of the 76-kDa fragment recognized by the N-terminal antibody is consistent with the size of the GST moiety, which was fused to the BLM N terminus (see Fig. 4A) and the
47-kDa N-terminal fragment produced in vivo. Western
analysis using anti-GST antibody confirmed that the 76-kDa fragment
contained GST (not shown). The cleavage patterns of native BLM (47 and
112 kDa) and GST-BLM (76 and 112 kDa) suggest that the caspase 3/7
cleavage site lies in the N-terminal third of the protein.
Identification of the Caspase Cleavage Site--
BLM contains only
a single four-amino acid cluster, 412TEVD415,
that is similar to the consensus caspase 3/7 recognition sequence DEVD
(11). The C-terminal aspartate in this cluster is located at amino acid
415 (Fig. 4A). Cleavage of native BLM at aspartate 415 would
yield two fragments with calculated molecular masses of 47 and
112 kDa, whereas cleavage of GST-BLM at this site would yield 112- and
76-kDa fragments (Fig. 4A). These predicted sizes match the
sizes of the BLM cleavage products generated in vivo and
in vitro.
To ascertain whether the sequence 412TEVD415 is
indeed the caspase recognition and cleavage site, we mutated aspartate
415 to glycine, generating the mutant protein BLM-D415G. Wild-type BLM
and BLM-D415G proteins were translated and radiolabeled in
vitro and then incubated with activated caspase 3. Wild-type BLM
was cleaved by caspase 3, whereas BLM-D415G was resistant to caspase 3 cleavage (Fig. 4B). This result suggests that caspase 3, or
possibly 7, cleaves BLM at aspartate 415 during apoptosis.
To determine whether caspase 3 or 7 cleaves BLM during apoptosis
in vivo, we used the caspase 3-deficient breast cancer cell line MCF-7 (51). MCF-7 cells were induced to undergo apoptosis by
TNF- The Cleaved N Terminus of BLM Is Dispensable for Helicase
Activity--
The 112-kDa apoptotic fragment contains the helicase and
DNA binding activities of BLM (Fig. 4A), but little is known about the
function of the smaller 47-kDa N-terminal fragment, which contains no
known protein motifs (52). To determine whether apoptotic cleavage,
which separates the N- and C-terminal regions, alters BLM helicase
activity, we incubated GST-BLM with activated caspase 3 in
vitro under conditions in which cleavage was complete (Fig. 3,
B and C). We then assayed intact and cleaved BLM
proteins for helicase activity. The substrate was a partial 20/46-mer
DNA duplex in which the 20-mer was radiolabeled at the 5'-end (Fig. 5A). Helicase activity
dissociates the 20-mer from the duplex; the duplex and dissociated
20-mer were distinguished using native PAGE and autoradiography, as
described (45).
Intact and cleaved GST-BLM completely unwound the helicase substrate
within 10 min (Fig. 5A, lanes 3, 4).
The activities of intact GST-BLM, GST-BLM cleaved by caspase 3, and
GST-BLM incubated with heat-inactivated caspase 3 were
indistinguishable at this time point (Fig. 5A, lanes
3-5), as well as earlier time points (see Fig. 5E).
Control reactions showed that the labeled 20-mer was released from the
duplex when heated (Fig. 5A, lane 1), whereas heat-inactivated GST-BLM, or GST treated with caspase 3, had no helicase activity (Fig. 5A, lanes 2,
6). Similar results were obtained when a longer partial
duplex DNA, or G4-DNA, were used as helicase substrates (not shown). We
conclude that BLM retains helicase activity after cleavage by caspase 3.
The C-terminal fragment retained helicase activity even after it was
physically separated from the N-terminal fragment. This was shown by
immobilizing GST-BLM on glutathione-Sepharose beads before incubating
with caspase 3. The C-terminal fragment was released into the
supernatant upon cleavage, whereas the N-terminal fragment containing
the GST moiety remained bound until eluted with glutathione (Fig.
5B). Western analysis confirmed that the C- and N-terminal
fragments were present in the appropriate fractions (Fig.
5B). The fragments, and intact protein, were tested
separately for helicase activity. The C-terminal fragment (Fig.
5C, lane 4), but not the N-terminal fragment
(lane 3), unwound the helicase assay substrate within 10 min, as did intact (lane 1) and cleaved (lane 2)
proteins. Control experiments using varying amounts of GST-BLM showed
that 2- to 5-fold differences in helicase activities could be detected
by the helicase assay (Fig. 5D, compare, for example, 5 versus 10 ng and 5 versus 25 ng) and that, under
assay conditions in which only a fraction of the substrate was unwound, uncleaved and cleaved BLM showed little or no difference in helicase activity.
These results suggest that the N-terminal 415 amino acids of BLM are
dispensable for helicase activity, and the C-terminal fragment
generated by apoptotic cleavage retains helicase activity.
Redistribution of BLM during Apoptosis--
Concomitant with
cleavage, apoptosis altered the subcellular localization of BLM. BLM is
found entirely in the nucleus, mostly, but not exclusively, organized
into discrete foci (Fig. 6A)
that costain for PML (31-34). BLM foci, detectable by indirect
immunofluorescence, began to disappear within 2 h after Jurkat
cells were induced to undergo apoptosis by anti-FAS (not shown).
Shortly thereafter (3-6 h after initiation of apoptosis) there was a
major redistribution of BLM. Much of the BLM was distributed outside
the areas of condensed DNA, which is visible by DAPI
fluorescence. Both the apoptotic BLM cleavage fragments,
detected by N-terminal (Fig. 6B) and C-terminal (Fig.
6C)-specific antibodies, showed this marked
redistribution.
Apoptotic cleavage of BLM very likely caused its release from the
nuclear matrix. Digitonin was used to permeabilize cytoplasmic and
nuclear membranes, and soluble, loosely bound proteins were separated
from insoluble cell structures by centrifugation (40). Western analysis
of the concentrated soluble (S) and insoluble (P) proteins showed that
a significant fraction of the BLM fragments detached from the insoluble
structures, whereas a small amount of full-length protein remained
bound (Fig. 6D). Because BLM is entirely nuclear and bound
to the nuclear matrix (31)2 and the insoluble fraction
contains nuclear and cytosolic matrix components, we infer that BLM
detaches from the nuclear matrix upon caspase cleavage. Because of the
fragility of cells undergoing apoptosis, it was not possible to obtain
standard nuclear matrix preparations, for example, by the methods
described by Wan et al. (53). Nonetheless, these results are
consistent with those obtained by immunofluorescence, suggesting that
BLM is cleaved and released from the nuclear matrix during apoptosis.
Caspases are important for both the initiation and execution
phases of apoptosis (7-9). At present, there is a need to identify caspase substrates to understand how caspases execute apoptosis. In
recent years, a number of caspase substrates have been identified (9,
54). Here, we show that BLM is a substrate for the execution caspase 3, in vitro and in vivo.
BLM encodes a DNA helicase (30) that is expressed primarily in late S
phase and G2 (55)2 and localizes to PML
nuclear bodies (31-34). The phenotypes associated with defects in BLM,
its homology to RECQ, and its colocalization with proteins such as
RAD51 and sites of repair after DNA damage2 suggest that
BLM participates in a homologous recombination repair pathway that
resolves spontaneous and/or induced DNA damage. The components of this
and other repair pathways likely exist in a large complex (56, 57). Our
finding that BLM is specifically cleaved in cells undergoing apoptosis,
but not necrosis, supports the idea that one function of the
execution caspases is to dismantle protein complexes that can repair
the DNA fragments generated by apoptotic deoxyribonucleases, as
proposed by Casciola-Rosen et al. (58).
In contrast to the cleavage of BLM, apoptosis did not result in
cleavage of the related RECQ-like helicase WRN. The WRN amino acid
sequence lacks consensus cleavage sites for caspases, suggesting that
WRN participates in processes that do not need to be dismantled during
apoptosis. Alternatively, WRN complexes may be targeted for disruption
by apoptosis, but one or more WRN-interacting proteins, rather than WRN
itself, may be subject to caspase cleavage.
Defects in WRN cause the Werner syndrome (WS). Although BS and WS have
similarities, there are also differences. Both syndromes are
characterized by a high incidence of cancer and cellular genomic instability (25, 26). However, WS individuals are asymptomatic until
after puberty, and survive much longer, generally until the fourth or
fifth decade of life. There are also differences between the BLM and
WRN proteins, despite similar helicase domains. WRN is not found in PML
nuclear bodies, and WRN, but not BLM, has intrinsic 3'-5' exonuclease
activity (45). BLM and WRN both exist in large complexes (56, 57).
However, the WRN complex contains many proteins that participate in DNA
replication, whereas the BLM complex contains many proteins that
participate in DNA damage sensing or repair. Components of the WRN and
BLM complexes may exchange, depending on the cell cycle or presence of
DNA damage, and there may be overlap in some functions of BLM and WRN.
However, because WRN and BLM associate primarily with different
complexes and nuclear structures, they may have different primary
functions. Interestingly, ATM, a component of the BLM complex, is also
cleaved during apoptosis (14). By contrast, Ku70, a component of
DNA-PK, was spared apoptotic cleavage, although the DNA-PKcs is cleaved (17, 58, 59). Ku was recently shown to interact with WRN (60). These
findings support the idea that apoptosis selectively targets processes
in which BLM, but not WRN, is a primary participant.
The major execution caspases are caspases 3, 6, and 7 (61, 62). GST-BLM
was efficiently cleaved by caspases 3 and 7, but not caspase 6, in vitro, and degradation in vitro was
indistinguishable from that observed in cells. It is not surprising
that both caspases 3 and 7 cleaved recombinant BLM, because these
enzymes share the same consensus cleavage recognition site (DEVD).
However, caspase 7 is localized predominantly to the endoplasmic
reticulum and mitochondria, whereas caspase 3 is found primarily in the
nucleus and cytoplasm (50). Our finding that BLM is not cleaved during apoptosis in the caspase 3-deficient cell line MCF-7 strongly implicates caspase 3, or one of its isoforms, rather than caspase 7, in
the apoptotic proteolysis of BLM in vivo.
Caspases cleave target proteins at specific sites, rather than
randomly, and cleavage may either activate or inactivate the substrate
(7-13). Proteolytic cleavage can provide information about the domain
structure of a protein, because protease-sensitive sites are often
interdomain regions that lack a defined secondary structure (63). We
mapped the region in BLM targeted by caspase 3 to a single site ~47
kDa from the N terminus. This site, 412TEVD415,
resembled the consensus caspase 3/7 recognition sequence DEVD (13). The
C-terminal BLM fragment generated by caspase 3 contains the helicase,
ATPase, DNA binding, and nuclear localization domains. The N-terminal
fragment is devoid of known protein motifs but was recently implicated
in assembling BLM into a hexamer (52, 64). DNA helicases are typically
dimers or hexamers (65). Oligomerization is thought to be essential for
processive translocation, and, in a few cases, oligomers are the active
form (65-68). Loss of the N-terminal fragment might prevent BLM
oligomerization but clearly did not abolish helicase activity, at least
in vitro. It is possible that domains outside the N-terminal
region can facilitate the assembly of BLM into hexamers or that the
C-terminal fragment is active as a monomer or other oligomeric form.
E. coli DNA helicase II is active as a monomer, although it
is capable of dimerization (69), and the WRN helicase appears to form
trimers (36). Finally, although loss of the N terminus did not abolish helicase activity in vitro, it might prevent hexamerization,
and hence activity, in vivo.
Whatever the biochemical outcome of caspase cleavage, immunostaining
and biochemical fractionation showed that cleaved BLM lost its
characteristic punctate nuclear localization, detached from an
insoluble substructure, and dissociated from condensed DNA. Cleavage
and loss of localization very likely obliterates the in vivo
function of BLM. BLM colocalizes with a number of proteins that are
essential for the repair of DNA by homologous recombination, including
RAD51 (56).2 RAD51 is also cleaved by caspase 3 during
apoptosis, and cleavage abolishes the RAD51 recombinase activity (15,
16). Moreover, the kinetics of BLM cleavage is similar to that of
DNA-PKcs (17, 58, 59), ATM (14), and PARP (18, 19), which are cleaved before CAD-induced DNA fragmentation occurs (10). The potential function of BLM in DNA repair suggests that its cleavage and
redistribution may aid nuclear disassembly and prevent the complex in
which it resides from participating in the repair of fragmented DNA
molecules generated by CAD.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5' DNA helicase that
localizes to the nuclear matrix and discrete nuclear foci known as PML or ND-10 bodies (25, 30-34). BLM foci also contain the
recombination/recombinational repair protein RAD51 and associate with
sites of putative DNA repair after damage by ionizing
radiation.2 These attributes
of BLM, and the cellular phenotypes of BS cells, suggest that BLM is
important for maintaining genomic integrity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Calbiochem) and 10 µg/ml cycloheximide (Sigma) or 2 µM staurosporine, and
MCF-7 cells were incubated with 30 ng/ml tumor necrosis factor-
and
10 µg/ml cycloheximide. To induce necrosis, Jurkat cells were washed
and suspended in serum-free medium lacking glucose and containing 2 mM pyruvate. After adaptation to the medium (37), they were
incubated with 2.5 µM oligomycin (Sigma). Pretreatment with 2.5 µM oligomycin was for 45 min, followed by
incubation for 6 h with or without anti-FAS. Intracellular ATP
levels were determined using a commercial kit (Sigma, FL-ASC) and
protocol furnished by the supplier. For protease inhibitor studies,
cells were preincubated with the indicated concentrations for 30 min, anti-FAS was added, and cells were harvested 6 h later, unless noted otherwise.
80 °C. Cell pellets were thawed on ice, suspended in 100 µl per
5 × 106 cells in buffer used by Dignam et
al. (38): 0.42 M NaCl, 10% glycerol, 20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride. The extract was clarified
by centrifugation at 10,000 × g for 15 min. Total cell
lysates were prepared by lysing 1-2 × 106 cells in
100 µl of SDS-PAGE sample buffer without dye. Protein concentrations
were determined by Bio-Rad detergent-compatible protein assay. Nuclear
extracts (30 µg) or total cell lysates (50 µg) were separated by
4-15% gradient (Bio-Rad) SDS-PAGE and analyzed by Western blotting as
described previously (39). Antibodies were detected by
chemiluminescence, using the SuperSignal West Pico detection kit (Pierce).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
BLM is cleaved in Jurkat cells undergoing
anti-FAS-induced apoptosis. Jurkat cells were treated with 100 ng/ml anti-FAS antibody to induce apoptosis and harvested at the
indicated times (0.5-12 h) thereafter. Nuclear extracts were prepared,
and 30 µg of protein was resolved by 10% SDS-PAGE and analyzed for
BLM, WRN, PARP, or Ku70 by Western blotting. Positions of the molecular
weight markers are indicated to the left of the
autoradiograms, and the approximate molecular weights of the intact
and cleaved proteins are indicated to the right.
A, BLM, detected by C-terminal BLM antibody; B,
WRN; C, Ku70; D, PARP.
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Fig. 2.
BLM is cleaved during apoptosis induced by
other stimuli. Cells were treated and harvested, as indicated.
Harvested cells were lysed in SDS-PAGE sample buffer, and analyzed for
BLM protein by Western blotting, using the anti-N-terminal
( -BLM NT) or C-terminal
(
-BLM CT) BLM antibody, as described under
"Experimental Procedures." The intact and cleaved BLM proteins are
indicated. A, HeLa cells were induced to undergo apoptosis
by addition of 30 ng/ml tumor necrosis factor-alpha (TNF)
and 10 µg/ml cycloheximide (CHX) and harvested 3 and
6 h thereafter. B, Jurkat (left panels) and
HeLa (right panels) cells were induced to undergo apoptosis
by addition of 2 µM staurosporine (STS) and
harvested at the indicated times (h) thereafter. C, Jurkat
cells were treated with 2.5 µM oligomycin (closed
circles), anti-FAS antibody (open circles), or ethanol
(solvent control) (open squares), all in glucose-free
medium. Lysates were prepared at the indicated times thereafter and
assayed for intracellular ATP as described under "Experimental
Procedures." ATP levels are shown as a percentage of level in
untreated cells. D, Jurkat cells were untreated
(Untreated), or induced to undergo necrosis using 2.5 µM oligomycin (Oligo) or apoptosis using
anti-FAS antibody (
-FAS), as described above.
Cells were harvested 6 h later. Cells were also pretreated with
oligomycin for 45 min, given anti-FAS antibody in the presence of
oligomycin (Oligo +
-FAS), and harvested
6 h later.
(TNF-
) and cycloheximide. Under these conditions, BLM cleavage was evident within 3 h, and near complete in 6 h (Fig. 2A). BLM was also cleaved 3-6 h after
either Jurkat or HeLa cells were induced to undergo apoptosis by the
protein kinase inhibitor staurosporine (Fig. 2B). In all
cases, BLM was cleaved to a 47-kDa fragment detected by the N-terminal
antibody and to a 112-kDa fragment detected by the C-terminal antibody (Fig. 2, A and B).
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Fig. 3.
BLM is cleaved by a caspase 3-like protease
during apoptosis. A, Jurkat cells were untreated
(Untreated, lane 1) or treated with anti-FAS
antibody ( -FAS, lane 2), and
harvested 6 h later. Alternatively, cells were preincubated with
the indicated protease inhibitors for 30 min, treated with anti-FAS in
the presence of inhibitors and harvested 6 h later. Lanes
3 and 4, 100 and 200 µM TLCK. Lanes
5 and 6, 100 and 200 µM leupeptin
(Leup). Lanes 7 and 8, 10 and 20 µM ZVAD-FMK. Lane 9, 100 µM
Ac-DEVD-CHO. Harvested cells were lysed and analyzed by Western
blotting using the anti-N-terminal BLM antibody. The intact and cleaved
BLM proteins are indicated. B, purified recombinant GST-BLM
(20 µM; Untreated) was cleaved for 1 h at
37 °C with 1 µM activated, recombinant caspase 3 (Caspase 3), 6 (Caspase 6), or 7 (Caspase
7). The cleavage products were analyzed by SDS-PAGE and Western
blotting, using the anti-N-terminal BLM antibody. The intact and
cleaved BLM proteins are indicated. C, GST-BLM was untreated
(Untreated) or cleaved with caspase 3 (Caspase
3), as described in B, and analyzed by SDS-PAGE and
silver staining to visualize the intact and cleaved BLM proteins, as
indicated.
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Fig. 4.
Identification of the BLM caspase cleavage
site. A, diagram showing GST-BLM, the GST and helicase
domains in BLM, and the GST-BLM and BLM cleavage products generated by
caspase 3. The caspase 3 recognition site,
412TEVD415, is indicated by the dark
vertical bar and asterisk. B, wild-type BLM
and the caspase-resistant mutant BLM-D415G were transcribed and
translated in vitro and digested with recombinant activated
caspase 3, as described under "Experimental Procedures." The intact
proteins and fragments were separated by SDS-PAGE and visualized by
autoradiography. C, MCF-7 cells were induced to undergo
apoptosis by tumor necrosis factor- (30 ng/ml) and cycloheximide (10 µg/ml). Total cellular lysates were prepared at the indicated
intervals (h) thereafter, and 50 µg of protein was analyzed by
Western blotting for BLM (N-terminal antibody) and PARP. The intact and
cleaved proteins are indicated.
and cycloheximide. As reported (51), partial cleavage of PARP
(Fig. 4C) and activation of caspase 7 (data not shown), which partially cleaves PARP in these cells, were apparent within 5 h. BLM, however, remained intact for at least 24 h (Fig.
4C). This result strongly suggests that caspase 3, not
caspase 7, cleaves BLM in vivo during apoptotic cell death.
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Fig. 5.
Helicase activity of intact and
caspase-treated BLM. Recombinant GST or GST-BLM (20 µM) were untreated or cleaved with activated recombinant
caspase (1 µM), and one-fourth the reaction mixture was
assayed for helicase activity using a 20-bp/46-bp partial duplex in
which the 20-bp oligonucleotide was 32P-labeled
(asterisk) at the 5'-end (illustrated to the left
of the upper band), as described under "Experimental
Procedures." Helicase activity released the single-stranded,
radiolabeled 20-mer (illustrated to the left of the
lower band). A, proteins were untreated
(lanes 2, 3), cleaved with caspase 3 (C3) (lanes 4-6), or proteins or probe were heat
denatured (H) (lanes 1, 2,
5) prior to assay, as described under "Experimental
Procedures." Lane 1, heat-denatured probe; lane
2, heat-denatured GST-BLM; lane 3, untreated GST-BLM;
lane 4, GST-BLM pretreated with caspase 3; lane
5, caspase 3 was heat-denatured prior to incubation with GST-BLM;
lane 6, GST pretreated with caspase 3. B, GST-BLM
was digested with caspase 3, and the reaction mixture was incubated
with glutathione beads. The beads were collected by centrifugation, and
the supernatant (Released) containing the released BLM
C-terminal fragment was recovered. The protein bound to the beads was
then eluted with glutathione, and the eluate was recovered
(Bound). Released and bound fractions were analyzed by
Western blotting using the anti-N-terminal ( -BLM
NT) or C-terminal (
-BLM CT) BLM antibody,
as described under "Experimental Procedures." The cleaved BLM
proteins are indicated. C, GST-BLM (lane 1),
GST-BLM digested with caspase 3 (lane 2), and the released
and bound fractions described in B were assayed for helicase
activity as described in A. D, GST-BLM (1-50 ng
as indicated) was assayed for helicase activity at room temperature for
30 min, as described under "Experimental Procedures." E,
GST-BLM (50 ng) was incubated at 37 °C for 1 h with buffer
(
Caspase) or 1 µM caspase 3 (+Caspase), and then assayed for helicase activity for the
indicated intervals (30-120 s) as described under "Experimental
Procedures." The unwound (Probe) and heat-denatured
(Probe, H) substrates are shown in the first two
lanes.
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Fig. 6.
Subcellular redistribution of cleaved
BLM. A-C, Jurkat cells were untreated
(A) or induced to undergo apoptosis using anti-FAS antibody
(B, C), fixed 3 h later, and stained for DNA
(DAPI) or BLM using either the anti-N-terminal
(A, B) or anti-C-terminal (C) BLM
antibody. Cells were viewed by epifluorescence microscopy, and the
fluorescent images were digitally merged (MERGE).
C, Jurkat cells were untreated (Control) or
induced to undergo apoptosis using anti-FAS antibody
(Apoptosis). Cells were extracted 4 h later with either
SDS-PAGE sample buffer (I, input) or 1%
digitonin. The digitonin extract was further separated into a soluble
supernatant (S) and insoluble pellet (P), the
proteins in each fraction were concentrated by precipitation and
denatured in SDS-PAGE sample buffer, as described under "Experimental
Procedures." Proteins were analyzed by Western blotting using
anti-N-terminal ( -BLM NT) or anti-C-terminal
(
-BLM CT) BLM antibodies, and anti-tubulin
antibody (Tubulin) as a control. Intact and cleaved BLM
proteins are indicated, as in Fig. 1A.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Ruth Lupu (Lawrence Berkeley National Laboratory) for the MCF-7 cells, Nathan Ellis (Sloan-Kettering Cancer Institute) for the N-terminal antibody, Guy Salvesen (The Burnham Institute) for recombinant caspases and expression constructs, Stephan Jackson (Cambridge University) for the C-terminal BLM antibody, Shurong Huang (Palo Alto Institute for Molecular Medicine) for the WRN antibody, and Scott Snipas (The Burnham Institute) for advice on expression of recombinant caspases.
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FOOTNOTES |
---|
* This work was supported by a Marie-Curie Fellowship (BMH4-CT98-5129 to O. B.), by National Institutes of Health Grants GM59901 (to T. K. S.) and AG11658 (to J. C. ), and by the U. S. Department of Energy under contract DE-AC03-76SF00098 to the University of California.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
Present address: Unite de Recombinaison et Expression
Genetique, Departement SIDA et Retrovirus, Institut Pasteur, 28 rue du
Docteur Roux, 75724 Paris, Cedex 15, France.
** To whom correspondence should be addressed: Lawrence Berkeley National Laboratory, Bldg. 84, Rm. 144, 1 Cyclotron Rd., Berkeley, CA 94720. Tel.: 510-486-4416; Fax: 510-486-4545; E-mail: jcampisi@lbl.gov.
Published, JBC Papers in Press, January 11, 2001, DOI 10.1074/jbc.M006462200
2 Bischof, O., Kim, S. H., Irving, J., Beresten, S., Ellis, N. A., and Campisi, J. J. Cell Bio. (in press).
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ABBREVIATIONS |
---|
The abbreviations used are:
CAD, caspase-activated deoxyribonuclease;
Ac-DEVD-CHO, acetyl-Asp-Glu-Val-Asp aldehyde;
BS, Bloom's syndrome;
DAPI, 4',6-diamidino-2-phenyl-indole;
DNA-PK, DNA-dependent
protein kinase;
DNA-PKcs, DNA-dependent protein kinase
catalytic subunit;
GST, glutathione S-transferase;
PAGE, polyacrylamide gel electrophoresis;
PARP, poly-ADP ribose polymerase;
PBS, phosphate-buffered saline;
PCR, polymerase chain reaction;
PML, promyelocytic leukemia protein;
TLCK, tosyl-L-lysine
chloromethyl ketone;
WS, Werner syndrome;
ZVAD-FMK, acetyl-Tyr-Val-Ala-Asp fluoromethyl ketone;
bp, base pair(s);
kb, kilobase(s);
TNF-, tumor necrosis factor-
.
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