Activation of the Apoptotic Endonuclease DFF40
(Caspase-activated DNase or Nuclease)
OLIGOMERIZATION AND DIRECT INTERACTION WITH HISTONE H1*
Xuesong
Liu,
Hua
Zou,
Piotr
Widlak
,
William
Garrard§, and
Xiaodong
Wang¶
From the Howard Hughes Medical Institute and Department of
Biochemistry and the § Department of Molecular Biology and
Oncology, University of Texas Southwestern Medical Center,
Dallas, Texas 75235
 |
ABSTRACT |
DNA fragmentation factor (DFF) is a heterodimeric
protein composed of 45-kDa (DFF45) and 40-kDa (DFF40) subunits, a
protein that mediates regulated DNA fragmentation and chromatin
condensation in response to apoptotic signals. DFF45 is a specific
molecular chaperone and an inhibitor for the nuclease activity of
DFF40. Previous studies have shown that upon cleavage of DFF45 by
caspase-3, the nuclease activity of DFF40 is relieved of inhibition.
Here we further investigate the mechanism of DFF40 activation. We
demonstrate that DFF45 can also be cleaved and inactivated by caspase-7
but not by caspase-6 and caspase-8. The cleaved DFF45 fragments
dissociate from DFF40, allowing DFF40 to oligomerize to form a large
functional complex that cleaves DNA by introducing double strand
breaks. Histone H1 directly interacts with DFF, confers DNA binding
ability to DFF, and stimulates the nuclease activity of DFF40 by
increasing its Kcat and decreasing its
Km.
 |
INTRODUCTION |
Chromatin condensation and DNA fragmentation into nucleosomal
fragments are the best recognized biochemical events of apoptosis (1,
2). Such events are mediated by the activation of
DFF,1 a heterodimeric protein
consisting of a 40-kDa (DFF40/CPAN/CAD) and a 45-kDa (DFF45/ICAD)
subunit (3-8). DFF45 is a dual function protein subunit that serves
both as a specific molecular chaperone to mediate the correct folding
of DFF40 and as an inhibitor of the DFF40 nuclease when complexed with
it (4, 6-8). DFF45 and its mouse homologue named ICAD are substrates
for caspase-3 and become cleaved in cells undergoing apoptosis (3, 4). The dual functions of DFF45 ensure that DFF will only become activated through the cleavage by caspases, the apoptotic proteases that only
become activated when cells receive apoptotic signals (9). In cell
extracts prepared from DFF45 knock out mice, DNA
fragmentation activity is completely abolished (8). In response to
apoptotic stimuli, splenocytes, thymocytes, and granulocytes from
DFF45 mutant mice are resistant to DNA fragmentation, and
splenocytes and thymocytes are also resistant to chromatin condensation
(8). Interestingly, unlike other nucleases, the activity of DFF40 can be markedly stimulated by the abundant chromatin-associated proteins such as histone H1, HMG-1, and HMG-2 (7, 10), which are known to be
located at the nucleosomal linker regions in chromatin (11, 12). We
have hypothesized that such a stimulatory effect facilitates the
generation of uniform nucleosomal fragments and also provides an
efficient way to disassemble complex chromatin structures (7).
In this communication, we report a novel mechanism for DFF40 activation
that involves DFF40 oligomerization and its direct interaction with
histone H1.
 |
EXPERIMENTAL PROCEDURES |
General Methods and Materials--
We obtained
[35S]methionine from Amersham Pharmacia Biotech, histone
H1 and anti-histone H1 antibody from Roche Molecular Biochemicals and
DNase I and micrococcal nuclease from Worthington. Plasmids were
purified using a Megaprep kit from Qiagen.
Production and Purification of Recombinant DFF--
The
expression plasmid containing both DFF45 and
DFF40 was constructed as described in Ref. 7 and transformed
into bacteria BL21(pLysS) (Novagen). DFF was induced and purified as
described in Ref. 7.
Production of Recombinant Caspase-3, -6, -7, and -8--
A
pET15b vector containing coding regions for caspase-3, caspase-6,
caspase-7, or a pET21a containing coding regions for
caspase-8 was used to transform bacteria BL21 (DE3). The
bacterial cultures (1 liter for each plasmid) were grown at 37 °C
until the density reached an A600 reading of
0.6. Isopropyl-1-thio-
-D-galactopyranoside was then
added to a final concentration of 2 mM. After a 3-h
induction, the bacteria were pelleted by centrifugation and lysed in
buffer A by sonication. After centrifugation, the supernatants were
loaded onto two 3-ml nickel-Sepharose (Qiagen) columns equilibrated
with buffer A. The columns were washed with 10 ml of buffer A (20 mM Hepes-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride) followed by 200 ml of
buffer A containing 500 mM NaCl and again with 10 ml of
buffer A. The fusion proteins were then eluted with buffer A containing
250 mM imidazole.
In Vitro Translation of Procaspase-3--
A pET 15b vector
(Novagen) containing a PCR fragment encoding amino acids 29-277 of
hamster caspase-3 was translated in a TNT T7
transcription/translation kit in the presence of
[35S]methionine according to the manufacturer's
instructions. The translated protein was passed through a 1-ml nickel
affinity column (Qiagen) equilibrated with buffer A. After washing the
column with 10 ml of buffer A, the translated caspase-3 was eluted with buffer A containing 250 mM imidazole.
Nuclease Assay for DFF--
An aliquot of recombinant DFF was
incubated with 60 ng of caspase-3 and 3 µg of pcDNA3 (Invitrogen)
at 37 °C for 30 min in a final volume of 30 µl adjusted with
buffer A containing 4 mM MgCl2. The reactions
were stopped by adding EDTA to a final concentration of 5 mM, and the samples were loaded onto 2% agarose gels
containing 2 µg/ml ethidium bromide. Electrophoresis was conducted at
50 V for 1 h in 0.5× Tris borate/EDTA buffer (1× Tris
borate/EDTA buffer contains 90 mM Tris borate, 2 mM EDTA). Gels were visualized under UV light.
Immunoprecipitation--
An aliquot of 1 ml of protein G-agarose
(Santa Cruz) was incubated with an aliquot of 200 µg of anti-histone
H1 antibody at 4 °C overnight. The protein G-agarose beads were
pelleted by centrifugation and washed five times with buffer A. The
beads were then incubated with 1 ml of bovine calf serum and 1 mg of
BSA for 1 h at room temperature. The beads were pelleted by
centrifugation and washed with buffer A five times. The beads were
resuspended in 1 ml of buffer A. Aliquots of 3 µg of DFF were
incubated alone, with 3 µg of histone H1, or with 3 µg of histone
H1 plus 5 µg of pcDNA3 in the absence or presence of 1 µg of
caspase-3 at room temperature for 30 min in a final volume of 300 µl
of buffer A followed by the addition of 50 µl of antibody protein
G-agarose beads prepared above. The samples were incubated at room
temperature for another hour, and the agarose beads were pelleted by
centrifugation and washed five times with buffer A. 60 µl of 1× SDS
loading buffer was added to the beads, and the samples were boiled at
100 °C for 3 min before being subjected to SDS-PAGE and Western blot analysis.
Immunoprecipitation with anti-DFF45 antibody was carried out by
incubating an aliquot of 500 µl of protein A-agarose with 500 µl of
preimmune or immune serum of DFF45 at 4 °C overnight. The antibody
protein A-agarose beads were pelleted by centrifugation and washed five
times with buffer A. The beads were then incubated with 1 ml of bovine
calf serum (1 mg/ml) for 1 h at room temperature, pelleted by
centrifugation, and washed with buffer A five times. The beads were
resuspended in 500 µl of buffer A. An aliquot of 50 µl of preimmune
protein A-agarose or immune protein A-agarose beads was incubated with
3 µg of DFF and 10 µg of histone H1 at room temperature for 1 h. The beads were harvested by centrifugation and washed five times
with buffer A. 60 µl of 1× SDS loading buffer was added to the
beads, and the samples were boiled at 100 °C for 3 min before being
subjected to SDS-PAGE and Western blot analysis.
Western Blot Analysis--
Polyclonal antibodies against DFF45
and DFF40 were produced as described (3, 7). A monoclonal antibody
against histone H1 was purchased from Roche Molecular Biochemicals.
Immunoblot analysis was performed with the horseradish
peroxidase-conjugated goat anti-mouse (histone H1) or goat anti-rabbit
(DFF45, DFF40) immunoglobulin G using enhanced chemiluminescence (ECL)
Western blotting detection reagents (Amersham Pharmacia Biotech).
 |
RESULTS |
Activation of DFF by Caspase-3 and -7--
To characterize the
enzymatic activity of DFF40, we generated recombinant DFF through a
bacterial double expression vector and purified the recombinant DFF to
apparent homogeneity through a nickel affinity column followed by a
Mono S column. Purified DFF exhibits a 1:1 ratio of DFF45 and DFF40,
which further proves that DFF40 and DFF45 form a stable complex (Fig.
1A).
It has been reported that in cells lacking caspase-3, DFF45 still gets
cleaved in response to apoptotic stimuli, suggesting that other
caspases may also cleave DFF45 (13, 14). Therefore we tested whether
apoptotic caspases other than caspase-3 could also activate DFF using
the purified recombinant DFF. As shown in Fig.
1B, both caspase-3 and
caspase-7 activated the nuclease activity of DFF (lanes 3 and 7). However, caspase-6 did not activate DFF (lanes
5 and 11), and caspase-8 only weakly activated DFF (lane 9). All these caspases did not show any nuclease
activity by themselves (lanes 2, 4, 6,
and 8). Although caspase-6 and -8 were unable to activate
DFF, these enzymes were indeed active as demonstrated by their ability
to cleave procaspase-3, a known substrate for these caspases
(lanes 11 and 12).

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Fig. 1.
Activation of DFF by different caspases.
A, the bacterial double expression vector (in pET15b) for
DFF (left) and a Coomassie Blue stained gel of purified
recombinant DFF (right). B, aliquots of 60 ng of
caspase-3, caspase-6, caspase-7, or caspase-8 were incubated with 3 µg of pcDNA3 in the presence or absence of 300 ng of DFF at
37 °C for 30 min in a final volume of 30 µl adjusted with buffer A
containing 4 mM MgCl2. The reaction samples
were loaded onto a 2% agarose gel containing 2 mg/ml ethidium bromide
(left). Aliquots of 60 ng of caspase-6 and caspase-8 were
incubated with 3 µl of in vitro translated,
[35S]methionine-labeled procaspase-3 at 37 °C for 10 min in 20 ml of buffer A (right). The samples were subjected
to 15% SDS-PAGE and transferred to a nitrocellulose filter. The filter
was exposed to a film for 16 h at room temperature.
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Oligomerization of DFF40 upon Activation by Caspase-3--
DFF
migrates on a gel filtration column as a heterodimer with a molecular
mass of ~85 kDa (3). When subjected to a Superdex 200 gel filtration
chromatography, the DFF peak was observed at fraction 15, corresponding
to the inactive heterodimeric form (Fig.
2A). The DNase activity of DFF
was readily detected when the column fractions were incubated with
caspase-3 (Fig. 2A, lower panel). Strikingly,
when DFF was activated by incubating with caspase-3 before being loaded
on the gel filtration column, a caspase-3-independent DNase activity
was now observed at fraction 10 after chromatography (Fig.
2B, lower panel), corresponding to a complex with
a size larger than the exclusion volume of this column (>1.3 million
daltons). This DNase activity co-migrated with one of the DFF40 peaks
as detected by Western blot analysis (Fig. 2B, middle
panel). The second DFF40 peak at fraction 16 showed no DNase
activity, indicating that only the large complex form of DFF40 is
active. The cleaved fragments of DFF45 now migrated at fractions
16-19, and no DNase activity was detected in these fractions. This
large DFF40 complex is responsive to histone H1 stimulation, and
including histone H1 during DFF activation does not interfere with the
formation of this large functional DFF40 complex (data not shown).

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Fig. 2.
Oligomerization DFF40 is required for
activation. In A, an aliquot of 5 µg of recombinant
DFF was fractionated by a Superdex-200 gel filtration column using a
Smart System (Amersham Pharmacia Biotech). Fractions of 100 µl were
collected. Aliquots of 30-µl column fractions were subjected to 12%
SDS-PAGE followed by Western blot analysis using a polyclonal antibody
against DFF40 (1:2000) and a polyclonal antibody against DFF45
(1:5000). The antigen-antibody complexes were visualized by an ECL
method as described under "Experimental Procedures." The filters
were exposed to x-ray film for 1 min (upper and middle
panels). In the lower panel, aliquots of 15-µl column
fractions were incubated with aliquots of 50 ng of bacteria-expressed
caspase-3, 300 ng of histone H1, 400 ng of BSA, and 500 ng of plasmid
DNA and MgCl2 at a final concentration of 4 mM
in 30 °C in a final volume of 25 µl in buffer A. After 1 h of
incubation, the samples were analyzed by a 2% agarose gel
electrophoresis and visualized by ethidium bromide staining. In
B, an aliquot of 5 µg of recombinant DFF complex was
preincubated with an aliquot of 200 ng of bacteria-expressed caspase-3
at 30 °C for 1 h in a final volume of 70 µl in buffer A. After incubation, the reaction mixture was fractionated in the gel
filtration column as in A. Aliquots of 30-µl column
fractions were subjected to 12% SDS-PAGE followed by Western blot
analysis as in A. The filters were exposed to x-ray film for
1 min (upper and middle panels). In the
lower panel, aliquots of 20-µl column fractions were
incubated with 300 ng of histone H1, 400 ng of BSA, and 500 ng of
plasmid DNA and MgCl2 at a final concentration of 4 mM at 30 °C in a final volume of 30 µl in buffer A. After 1 h of incubation, the samples were analyzed as in
A.
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Interaction of Histone H1 and DFF--
Previously, we have shown
that the endonuclease activity of DFF40 can be stimulated by
chromatin-associated proteins such as histone H1 and HMG proteins (7,
10). To elucidate the mechanism of this stimulatory effect, we tested
whether histone H1 could directly interact with DFF. The recombinant
DFF was incubated with histone H1 in the absence (Fig.
3A, lanes 1-3) or
presence (lanes 4-6) of caspase-3. The protein complexes
were immunoprecipitated with a monoclonal anti-histone H1 antibody and
analyzed by Western blotting using antibodies against DFF45, DFF40, and
histone H1 (Fig. 3A). When caspase-3 was not included in the
reaction, both DFF40 and DFF45 were found to co-precipitate with
histone H1 (Fig. 3A, lane 2). However, in the
presence of caspase-3, only DFF40 was co-precipitated with histone H1
(Fig. 3A, lane 4). Inclusion of DNA in the
reaction did not increase the binding between DFF and histone H1 (Fig.
3A, lanes 3 and 6). As a control, DFF
alone could not be precipitated by the anti-histone H1 antibody (Fig. 3A, lanes 1 and 4). Furthermore, the
cleaved fragments of DFF45 did not co-precipitate with histone H1 and
resided exclusively in the supernatant as detected by Western blot
analysis (data not shown).

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Fig. 3.
Histone H1 directly interacts with DFF40.
A, aliquots of 3 µg of DFF were incubated alone
(lanes 1 and 4), with 3 µg of histone H1
(lanes 2 and 5), or with 3 µg of histone H1
plus 5 µg of pcDNA3 (lanes 3 and 6) in the
absence (lanes 1-3) or presence (lanes 4-6) of
1 µg of caspase-3 at room temperature for 30 min in a final volume of
300 µl adjusted with buffer A. In A, aliquots of 70 µl
of protein G-agarose coupled with anti-histone H1 antibody were
included in each reaction (left). After 1 h of
incubation at room temperature, the agarose beads were precipitated and
washed five times with 500 µl of buffer A. 60 µl of 1× SDS loading
buffer was added to the agarose beads and heated at 100 °C for 3 min. After spinning at 14,000 rpm in a microcentrifuge for 1 min,
aliquots of 20 µl of the resulting supernatant were subject to
SDS-PAGE, and Western blot analysis was carried out using anti-DFF45,
anti-DFF40, or anti-histone H1 antibody, and the antigen-antibody
complexes were visualized by an ECL method as described under
"Experimental Procedures." The filters were exposed to x-ray film
for 1 min. In A, an aliquot of 3 µg of DFF was incubated
with 10 µg of histone H1 at room temperature for 30 min in a final
volume of 300 µl adjusted with buffer A (right). Then
aliquots of 50 µl of protein A-agarose beads coupled with either the
preimmune serum (lane 7) or the anti-DFF45 serum (lane
8) were added to half of the reaction mixture, and the incubation
was continued for 1 h. The agarose beads were washed five times
with 500 ml of buffer A. 60 µl of 1× SDS loading buffer was added to
the agarose beads and heated at 100 °C for 3 min. Aliquots of 20 µl were subject to SDS-PAGE, and Western blot analysis was carried
out using anti-DFF45, anti-DFF40, or anti-histone H1 antibody. In
B, two primers were used to PCR amplify a 1-kilobase DNA
fragment containing the DFF45 coding region in the absence or presence
of [32P]dATP. The PCR products were purified by passing
through a PCR purification column (Qiagen). An aliquot of 3 µg of the
unlabeled PCR product was mixed with 100,000 cpm of the same
32P-labeled PCR product and incubated with the indicated
amounts of DFF (60, 180, 600, and 1800 ng) in the absence or presence
of 3 µg of histone H1 at room temperature for 30 min. Then 50 µl of
the DFF45 antibody-coupled protein A-agarose was added to each
reaction. After 30 min of incubation at room temperature, the agarose
beads were pelleted by centrifugation and washed five times with 1 ml
of buffer A. The radioactivity in the agarose beads was detected by
scintillation counting. C, aliquots of 1.5 µg of DFF were
incubated with 1.0 µg of caspase-3 at 37 °C for 5 min with a final
volume of 100 µl of buffer A. Then aliquots of various amounts of
sheared DNA (10, 15, 25, 40, 50, and 65 µg) were added to each
reaction in the absence or presence of 10 µg of histone H1 with a
final volume of 0.5 ml of buffer A containing 4 mM
MgCl2. The absorbances of the reaction mixtures at 260 nm
(A260) were recorded at different times, and the
slope of the linear increase on the A260 was
defined as the initial velocity of the reaction. The concentration of
DNA (g/liter) in the reaction was defined as substrate
concentration(s). The Kcat and
Km values were determined by plotting 1/velocity
against 1/substrate concentration.
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To confirm such interaction between histone H1 and DFF, we also carried
out the reciprocal immunoprecipitation experiment using an antiserum
against DFF45. Both DFF40 and histone H1 were co-precipitated by the
anti-DFF45 antibody (Fig. 3A, lane 8). In
contrast, the preimmune serum did not precipitate either protein (Fig.
3A, lane 7).
Because DFF is located in the nucleus (7, 15), it was of interest to
see whether DFF could directly bind to DNA. We tested DNA binding by
conducting an immunoprecipitation experiment using the anti-DFF45
antibody. As shown in Fig. 3B, in the absence of histone H1,
very little DNA was co-precipitated even with the highest amount of DFF
used. However, in the presence of histone H1, significant amounts of
DNA were co-precipitated with DFF in a dose-dependent fashion.
To evaluate the effect of histone H1 on the kinetics of nuclease
activity of DFF, we measured the nuclease activity of DFF in the
absence or presence of histone H1 using different concentrations of
sheared DNA as substrates. The velocities of the reactions were
determined by the Kunitz method (16). As shown in Fig. 3C,
histone H1 causes a 2-fold increase on Kcat and
a 4-fold decrease on Km of the nuclease activity of
DFF.
DFF Cleaves DNA by Introducing Double Strand Breaks--
To
determine how DFF cleaves double-stranded DNA, we compared the cleavage
products of DFF with that of DNase I by two-dimensional gel
electrophoresis. As shown on Fig.
4c, after two-dimensional alkaline electrophoresis, a horizontal distribution of single-stranded DNA was observed for DNase I digestion, confirming that DNase I
introduces single strand nicks into double-stranded DNA (17). However,
the DNA cleaved by DFF showed a diagonal distribution on
two-dimensional gel electrophoresis that was different from the pattern
generated by DNase I (Fig. 4, a and c),
indicating that DFF did not generate single strand nicks on
double-stranded DNA. Inclusion of histone H1 in the reaction did not
change the diagonal distribution of cleavage products on the
two-dimensional gel (Fig. 4b), suggesting that histone H1
stimulates the nuclease activity of DFF without changing its mode of
DNA cleavage.

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Fig. 4.
DFF40 cleaves DNA by introducing
double-stranded DNA breaks. 3 µg of pcDNA3 and 150 ng of
caspase-3 were incubated with 100 ng of DFF (a) or 20 ng of
DFF plus 0.5 µg of histone H1 (b) at 37 °C for 15 min
in a final volume of 40 µl adjusted with buffer A containing 4 mM MgCl2. In c, 3 µg of pcDNA3
were incubated with 0.05 unit of DNase I at 37 °C for 5 min in a
final volume of 30 µl adjusted with buffer B (50 mM
Tris-HCl, pH 8.0, 1 mM MgCl2, and 1 mM CaCl2). Samples were run in 0.5× TAE (40 mM Tris-acetate, 1 mM EDTA) agarose gel
(1st dimension, neutral). After treatment with denaturing
buffer (50 mM NaOH, 1 mM EDTA), the gels were
turned 90° and run in the same buffer (2nd dimension,
alkaline).
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 |
DISCUSSION |
Caspases have been suggested to be the core apparatus of the
execution of cell death (9). The cleavage of DFF45 by caspase-3, which
liberates the nuclease activity of DFF40, supports this suggestion. In
addition to caspase-3, DFF can also be activated by caspase-7 and
weakly activated by caspase-8 but not by caspase-6 (Fig.
1B). This is consistent with previous studies on the
substrate preferences of caspases, with caspase-3 and caspase-7
preferring the DXXD motif that fits the caspase cleavage
sites DETD and DAVD on DFF45. On the other hand, caspase-6, -8, and -9 prefer the (L/V)EXD motif that is absent in DFF45 (18,
19).
The cleavage of DFF45 by caspases, interestingly, does not just
dissociate the fragments of DFF45 from DFF40 to release its inhibition;
in addition, liberated DFF40 oligomerizes to form a large protein
complex that elutes at the exclusion volume of a Superdex 200 gel
filtration column (Fig. 2). This large DFF40 oligomer is by itself an
active DNase and is responsive to histone H1 stimulation. The formation
of this large DFF40 oligomer probably requires the chaperoning function
of DFF45 because the expression of DFF40 alone cannot generate
functional DNase both in cultured cells and in DFF45 knock
out mice (4, 6-8).
Purified DFF exhibits relatively weak nuclease activity in the presence
of caspase-3, whereas the same amount of protein can actively induce
DNA fragmentation in nuclei (3). Based on this observation, we have
identified additional protein factors, such as histone H1 and HMG-1/-2,
that enhance the nuclease activity of DFF (7, 10). Histone H1 and HMG
proteins are "architectural" proteins that are located in the
internucleosomal linker regions of chromatin (11, 12). The association
of DFF with these chromatin-associated proteins therefore targets DFF
to the linker regions. Moreover, the direct interaction of DFF with
histone H1 also confers the DNA binding ability to DFF (Fig.
3B).
Previously, we have shown that histone H1 stimulates the nuclease
activity of DFF more than 10-fold (7). Kinetic analysis indicates that
histone H1 enhances the nuclease activity of DFF by increasing
Kcat and decreasing Km (Fig.
3C). We postulate that HMG proteins may have a role similar
to histone H1. Of the HMG proteins tested, HMG-1, HMG-2, and HMG-14 but
not HMGI/Y stimulated the nuclease activity of DFF (data not shown).
One possibility is that HMGI/Y does not interact directly with DFF
although histone H1 and HMG-1, -2, and -14 do. Therefore histone H1 and
HMG proteins not only recruit DFF to the internucleosomal linker
regions of chromatin but also stimulate the nuclease activity of DFF40
oligomer. Recently, Halenbeck et al. (6) stated that carrier
proteins such as BSA also enhance the endonuclease activity of
DFF40/CPAN in the presence of higher concentrations of
Mg2+. However, our results indicate that low concentrations
of BSA have no effect on DFF40 nuclease activity (7), and only when high concentrations of BSA (1 mg/ml) were included in the reaction buffer did the BSA effect become obvious (data not shown). The stimulatory effect of histone H1 on DFF40 nuclease is at least two
orders of magnitude higher than that of BSA (data not shown). Furthermore, histone H1 and HMG-1/-2 still stimulated the nuclease activity of DFF40 when we used the same assay conditions as described by Halenbeck et al. (6) (data not shown). Therefore the
stimulatory effect of chromatin-associated proteins on DFF is different
from that of BSA.
Unlike DNase I, DFF40 did not generate single strand nicks when
cleaving double-stranded DNA (Fig. 4), indicating that DFF40 cleaves
DNA by introducing double strand breaks. The break points are either
blunt ends or one-base 5'-overhangs with 5'-phosphate and 3'-hydroxyl
groups (data not shown). Such a property eliminates the possibility for
single strand repair enzymes to relegate the broken points. In
addition, because double-stranded DNA breaks are themselves apoptotic
signals, the cleavage of genomic DNA by DFF40 may trigger an
amplification cycle that marks an irreversible step for apoptosis.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Emad Alnemri at Thomas Jefferson
University for the expression construct of caspase-8 and caspase-9, Dr.
Michael Bustin at National Institutes of Health for recombinant HMG14, Dr. Raymond Reeves at Washington State University for recombinant HMGI/Y, and Dr. R. G. Johnson at UCLA for purified HMG-1 and
HMG-2.
 |
FOOTNOTES |
*
This work was supported in part by Grant RE258 from the
American Cancer Society, Grant GMRO1-55942 from the National Institutes of Health, Grant I-1412 from the Robert Welch Foundation (to X. W.),
Grants GMRO1-29935 and GMRO1-51585 from the National Institutes of
Health, and Grant I-823 from the Robert Welch Foundation (to W. G.).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.
On leave from the Institute of Oncology, Gliwice, Poland.
¶
To whom correspondence should be addressed: HHMI, Dept. of
Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235. Tel.: 214-648-6713; Fax:
214-648-5419; E-mail: xwang{at}biochem.swmed.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
DFF, DNA
fragmentation factor;
DFF45, 45-kDa subunit of DFF;
DFF40, 40-kDa
subunit of DFF;
CAD, caspase-activated DNase;
ICAD, inhibitor of CAD;
CPAN, caspase-activated nuclease;
PAGE, polyacrylamide gel
electrophoresis;
PCR, polymerase chain reaction;
BSA, bovine serum
albumin;
HMG, high mobility group.
 |
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