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INTRODUCTION |
Poly(ADP-ribose) polymerase
(PARP)1 is a highly abundant
nuclear enzyme present at about 2 × 105 molecules per
nucleus (1). This enzyme is composed of an N-terminal DNA binding
domain, containing two zinc finger motifs, a C-terminal NAD+ binding domain, catalyzing the synthesis of ADP-ribose
polymers from its substrate, NAD+, and an automodification
site, which unites the N-terminal and C-terminal domains (2).
Poly(ADP-ribosyl)ation by PARP at the automodification site of the
protein is initiated by the binding of the zinc fingers to DNA breaks
(3, 4). As a consequence of this automodification, the binding affinity
of PARP for DNA is reduced, resulting in dissociation of PARP from DNA
breaks (5) and thereby allowing the DNA repair machinery to access the
sites of DNA damage (6).
In cells where DNA breaks are generated by DNA-damaging agents, PARP is
activated and automodified (3, 4), leading to the conclusion that PARP
is involved in the cellular response to genetic damage, particularly in
the repair of damaged DNA (3). However, PARP has been shown to lack DNA
repair activity in itself (6, 7). Alternatively, it has been suggested
that PARP is involved in chromatin stabilization (8), in DNA
replication (9, 10), and in transcription (11-13), although the roles played by PARP in these processes are not yet understood.
In nuclear localization experiments, PARP is observed in clear foci,
associated both with regions of chromatin actively transcribed by RNA
polymerase II as well as with nucleoli where rRNA is synthesized by RNA
polymerase I (14). Dispersal of the foci upon treatment of cells with
the transcription inhibitors actinomycin D or
5,6-dichloro-1-
-ribofuranosylbenzimidazole suggests an involvement
of PARP in transcription (15). In addition, such foci are also
dispersed by treatment of isolated nuclei with RNase (16). These
observations thus suggest an interaction between PARP and transcribed
RNA. Recently, we demonstrated that RNA-bound PARP reduces the rate of
RNA elongation by RNA polymerase II and that automodification of PARP
in response to DNA damage up-regulates transcription (17). Since
DNA-damaging agents induce RNA damage as well, we proposed that this
up-regulation allows cells to compensate for the loss of damaged RNA
that occurs collaterally with DNA damage and that this pathway is
required for cell survival following exposure to DNA-damaging agents
(17).
When cells are exposed to sufficiently high levels of DNA-damaging
agents, they commit to cell death by inducing either apoptosis or
necrosis. During apoptosis PARP is cleaved by the apoptosis-specific protease, caspase-3, resulting in the formation of an N-terminal 24-kDa
fragment, containing the DNA binding domain, and a C-terminal 89-kDa
catalytic domain, containing the automodification site (18-20).
Recently, Halappanavar et al. (21) and Oliver et
al. (22) reported that, in PARP knockout cells, expression of
uncleavable PARP, lacking the caspase-3 recognition sequence, causes
delayed induction of DNA damage-induced apoptosis. This observation
suggests that cleavage of PARP has a role in damage-induced apoptosis. In addition, Herceg and Wang (23) and Boulares et al. (24) suggested that cleavage of PARP is also required for tumor necrosis factor-
-induced cell death. However Herceg and Wang (23) found increased cell death by necrosis, whereas Boulares et al.
(24) demonstrated a promotion of apoptotic, rather than necrotic, cell death by expressing uncleavable PARP in PARP knockout cells.
Since the 24-kDa fragment contains the DNA binding domain, which is
capable of binding to DNA breaks (25), it has been speculated that the
24-kDa fragment possibly counteracts functions of PARP and promotes the
process of apoptosis (19). However, biochemical characteristics of the
24-kDa fragment remain to be elucidated. Thus, we prepared the 24-kDa
apoptotic fragment of PARP and asked whether the 24-kDa fragment
competes against the functions of PARP in DNA repair, ADP-ribose
polymer formation, and transcription.
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MATERIALS AND METHODS |
Cell Line and Antibody--
GMO1953A lymphoblastoid cells were
obtained from NIGMS Human Mutant Cell Repository (Camden, NJ). The
C-II-10 antibody against the automodification domain of PARP and the
F1-23 antibody against the DNA binding domain of PARP (zinc finger 2)
(26) were kindly provided by Dr. G. G. Poirier.
Expression of Recombinant PARP and the 24-kDa
Fragment--
Full-length PARP cDNA (bases 1-3039) or the
sequence corresponding to the 24-kDa DNA binding domain of PARP (bases
1-654), which is found in apoptotic cells (18-20), was cloned into
pET3a (Novagen) and used to transform HMS 174 de3 cells (Novagen)
together with pLysE (Novagen). After overnight pre-culture, the
Escherichia coli were propagated in 2 liters of
Luria-Bertani medium in the presence of 34 µg/ml chloramphenicol and
100 µg/ml ampicillin for 3 h, and expression of PARP or the
24-kDa fragment was induced in the presence of 0.4 mM
isopropyl-
-D-thiogalactoside for 3 h at 37 °C.
The bacteria were then spun down at 3500 × g for 10 min, and the pellet was washed in phosphate-buffered saline and spun
down. The resulting pellet was resuspended in 20 ml of buffer containing 0.1 M NaCl, 50 mM Tris-HCl, pH
8.0, 12% glycerol, 2 mM MgCl2, and 0.1 mM phenylmethylsulfonyl fluoride (Buffer CB), and PARP or
the 24-kDa fragment was extracted by sonication. After a 30-min
centrifugation at 35,000 × g (at 4 °C), the
supernatant was used for purification of PARP or the 24-kDa fragment.
Purification of PARP--
E. coli lysate (750 mg) was applied to a phosphocellulose column (10 mm diameter and 2-ml
bed volume) equilibrated with Buffer CB. PARP was eluted using a linear
gradient of Buffer CB containing 0.1-2.0 M NaCl. The
fractions of interest were determined by silver stain analysis of
samples run on SDS-12.5% polyacrylamide gels. The fractions of
interest were then pooled, diluted with Buffer CB to reduce the NaCl
concentration below 0.2 M, and applied to a DNA cellulose
column (5 mm diameter and 2-ml bed volume) equilibrated with Buffer CB.
After washing the column with 20 ml of Buffer CB followed by 8 ml of
Buffer CB containing 0.4 M NaCl, PARP was eluted with 8 ml
of Buffer CB containing 1.0 M NaCl. The eluate was diluted
with Buffer CB to reduce the NaCl concentration below 0.2 M
and applied to a heparin-Sepharose column (Amersham Pharmacia Biotech,
Hi-Trap, 5 ml) equilibrated with Buffer CB. PARP was eluted using a
linear gradient of Buffer CB containing 0.1-2.0 M NaCl,
and the final fractions were dialyzed against Buffer CB prior to analysis.
Purification of the 24-kDa Fragment--
Purification was
carried out as for PARP except that the DNA cellulose chromatography
was omitted. The final preparation was dialyzed against Buffer CB prior
to analysis.
Quantitation of PARP and the 24-kDa Fragment by Direct
ELISA--
The purified recombinant PARP and the 24-kDa fragment were
quantified by direct ELISA. Briefly, PARP and the 24-kDa fragment were
immobilized on a microtiter plate, treated with the antibody F1-23
(26) (5000-fold dilution) which recognizes the zinc finger 2 motif
present in both PARP and the 24-kDa fragment, and then treated with
secondary antibody conjugated to horseradish peroxidase. Following
addition of TMB substrate (Research Diagnostics Inc.), 0.18 M H2SO4 was used to initiate the
peroxidase reaction. PARP and the 24-kDa fragment were quantified based
on absorbance at 490 nm.
Gel Retardation Assay (DNA)--
To prepare the DNA probe for
the gel retardation assay, a single-stranded 50-base
oligodeoxynucleotide (100 pmol) was 32P-labeled at 37 °C
for 15 min using 25 µCi of [
-32P]ATP with 10 units
of T4 polynucleotide kinase (Amersham Pharmacia Biotech) in a 25-µl
reaction mixture using the buffer provided by the supplier. The labeled
single-stranded oligodeoxynucleotide was annealed with its
complementary single-stranded oligodeoxynucleotide (50 bases) in a
100-µl mixture containing 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA by incubating for 5 min at 95 °C, followed by 5 min at 50 °C, and then 20 min at 22 °C. The annealed
double-stranded oligodeoxynucleotide was then precipitated with ethanol
and ammonium acetate, and the resulting pellet was dissolved in 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA. The binding
reaction was carried out using 1 pmol of the 32P-labeled
double-stranded oligodeoxynucleotide with varying amounts of PARP or
the 24-kDa fragment in a buffer containing 5 mM Tris-HCl, pH 8.0, and 5 mM MgCl2 for 15 min at 30 °C
in a 15-µl reaction mixture. Samples were then fractionated by native
6% polyacrylamide gel electrophoresis, and the gel was dried and
exposed to x-ray film for autoradiography or used for quantitation by
AlphaImager (Packard Instrument Co.).
Gel Retardation Assay (RNA)--
Gel retardation assays using an
RNA probe were carried out as described previously (17). Briefly,
32P-labeled stem-loop RNA was synthesized using T3 RNA
polymerase to transcribe the human immunodeficiency virus, type I TAR
sequence. The 32P-labeled stem-loop RNA was then incubated
with PARP or the 24-kDa fragment and fractionated by native 6%
polyacrylamide gel electrophoresis. After drying, the gel was either
exposed to x-ray film for autoradiography or used for quantitation by
AlphaImager (Packard Instrument Co.).
Cell-free DNA Repair Assay--
Cell-free extracts were prepared
from GMO1953A lymphoblastoid cells following the method of Manley
et al. (27). The cell-free DNA repair assay was carried out
using 50 µg of extract, 300 ng of
-irradiated pBluescript II
KS+ (pBS, 3 kilobase pairs) containing an average of one
single-stranded DNA break per molecule (6), and varying amounts of the
24-kDa fragment in the presence or absence of 2 mM
NAD+ under reaction conditions described previously (28).
After purification of the DNA, unrepaired pBS (open circular) and
repaired pBS (closed circular) were resolved by ethidium bromide, 1%
agarose gel electrophoresis.
Analysis of Poly(ADP-ribosyl)ation--
To determine the amount
of ADP-ribose polymers produced in the cell-free DNA repair assay, 1.3 µCi of [32P]NAD+ and 0.25 mM
NAD+ were added to the reaction described above. Reactions
were terminated by addition of trichloroacetic acid, and insoluble
32P activity retained on a GF/C filter was counted as
described previously (28).
Pulse-Chase Elongation Assay--
Pulse-chase elongation
assays were carried out as described previously (17). Briefly, a pGf1
plasmid containing a G-less sequence was digested with PacI,
creating a 90-base G-less sequence at one end of the DNA. A C-tail was
added to allow loading of RNA polymerase II from DNA break ends. The
DNA was then incubated with RNA polymerase II (1.0 units) (provided by
Dr. H. Serizawa) (29) in the presence of RNasin, ATP, UTP, CTP, and
[
-32P]CTP at 37 °C for 30 min as described by
Shilatifard et al. (30). A chase was initiated by addition
of GTP and excess CTP in the presence or absence of PARP or the 24-kDa
fragment. After extraction and precipitation, RNA was fractionated on a
6% polyacrylamide, 8 M urea gel. After drying, the gel was
exposed to x-ray film for autoradiography.
Cell-free Transcription Assay--
pCMV-Luc containing the
cytomegalovirus promoter was linearized by ScaI restriction
digestion and used in a run-off transcription assay with HeLa nuclear
extracts (Promega). The expected size of the run-off product was 400 bases. Reactions were carried out for 1 h at 30 °C using 4 µg/ml linearized pCMV-Luc in a reaction mixture containing 10 mM HEPES-KOH, pH 7.9, 40 mM KCl, 0.2 mM dithiothreitol, 3 mM MgCl2, 400 µCi/ml [
-32P]GTP, 400 µM ATP, 400 µM CTP, 400 µM UTP, 16 µM
GTP, 10% glycerol, 1000 transcription units/ml of HeLa nuclear
extract, and varying amounts of either PARP or the 24-kDa fragment. The
reaction was terminated by addition of 7 volumes of 0.3 M
Tris-HCl, pH 7.4, 0.3 M sodium acetate, 0.5% SDS, 2 mM EDTA, and 3 µg/ml tRNA. Following phenol/chloroform
extraction, transcripts were precipitated with ethanol, fractionated by
3% polyacrylamide, 8 M urea gel electrophoresis, and
visualized by autoradiography.
Apoptosis Induction and TUNEL Assay--
The mammalian
expression vector pcDNA 3.1
(Invitrogen) was used to clone the
cDNA corresponding to the 24-kDa DNA binding domain of PARP
(pcDNA 3.1
/AF24). Then 1.25 µg/ml pcDNA3.1-/AF24 was
incubated with 3.75 µl/ml Tfx20TM reagent (Promega) in
serum-free Dulbecco's modified Eagle's medium (serum-free DMEM) for
10 min at room temperature and used to treat HeLa S3 cells (attached to
coverslips) for 1 h at 37 °C. Following cell treatment, DMEM containing 10% fetal bovine serum and antibiotics (complete DMEM) was
added; HeLa S3 cells were cultured for 24 h after which the medium
was replaced with serum-free DMEM, and the cells were exposed to
N-methyl-N'-nitro-N-nitrosoguanidine
(MNNG) (50 µM). After 20 min of treatment at 37 °C,
the medium was replaced with complete DMEM, and cells were cultured for
2 h. TUNEL staining (fluorescein in situ cell death
detection kit, Roche Molecular Biochemicals) was then carried out after
fixing the cells in 3% paraformaldehyde and permeabilizing them with
0.1% Triton X-100 and 0.1% sodium citrate according to the
supplier's instructions (Roche Molecular Biochemicals). TUNEL-positive
cells were visualized by fluorescence microscopy.
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RESULTS |
Recombinant PARP and the 24-kDa Fragment--
To investigate the
effect of the 24-kDa PARP fragment on DNA repair and transcription, we
first prepared the recombinant 24-kDa fragment and full-length PARP. As
shown in Fig. 1, the 24-kDa fragment,
which migrated to an apparent molecular mass of about 30 kDa on
SDS-polyacrylamide gels, was purified to over 99% homogeneity as
described under "Materials and Methods". The purity of full-length recombinant PARP was about 95%, with several truncated products observed (Fig. 1). Quantitation of the 24-kDa fragment and PARP was
carried out by direct ELISA using the F1-23 antibody (26), which
recognizes the zinc finger 2 motif of PARP and the 24-kDa fragment.

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Fig. 1.
Purified recombinant 24-kDa fragment and
PARP. Recombinant 24-kDa fragment and PARP were purified as
described under "Materials and Methods," fractionated by SDS-12.5%
polyacrylamide gel electrophoresis, and visualized by silver
staining.
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Binding of the 24-kDa Fragment and PARP to DNA
Breaks--
32P-Labeled double-stranded
oligodeoxynucleotide (50 base pairs) was incubated with either the
24-kDa fragment or PARP. If the zinc finger motifs found in PARP and
the 24-kDa fragment bind to DNA ends (2, 31), the mobility of the
32P-labeled DNA probe should be reduced on a native gel. As
shown in Fig. 2, discrete retarded bands
were in fact observed when the labeled probe was incubated with the
24-kDa fragment. Migration of the probe was similarly retarded when
PARP was used instead of the 24-kDa fragment, with the probe migrating
slightly more slowly than in the case of the 24-kDa fragment. In
addition, the labeled probe was reproducibly found at the origin of the
lane. Addition of excess unlabeled double-stranded oligodeoxynucleotide inhibited retardation of the probe, confirming that formation of the
retarded labeled material was due to binding of the 24-kDa fragment or
PARP to the DNA probe (data not shown). The data in Fig. 2 reveal a
linear relationship between 32P activity associated with
the retarded fractions and the amount of the 24-kDa fragment or PARP
and allow us to determine that the 24-kDa fragment has about 25% of
the binding activity of full-length PARP. Thus, even after cleavage of
PARP by caspase-3, the resulting 24-kDa fragment retains significant
DNA binding activity.

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Fig. 2.
Gel retardation assay (DNA). Recombinant
24-kDa fragment or PARP was incubated with 32P-labeled
50-base pair double-stranded DNA probe, and the DNA was then
fractionated on a native 6% polyacrylamide gel. The 32P
activity was visualized by autoradiography, and the 32P
activity in the retarded fraction (including origin) was quantified
with InstantImager (Packard Instrument Co.); relative activity is shown
(arbitrary units).
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Inhibition of DNA Repair by the 24-kDa Fragment--
In the
absence of PARP's substrate, NAD+, PARP binds to and
persists on DNA breaks, thereby inhibiting DNA repair (6). Thus, dissociation of PARP from DNA breaks by automodification is a prerequisite for DNA repair (6). Since the 24-kDa fragment is capable
of binding to DNA breaks (Fig. 2) but lacks the automodification site,
these fragments should persist on DNA breaks and inhibit DNA repair
even in the presence of NAD+. To test this hypothesis, a
cell-free DNA repair assay was carried out using open circular pBS
containing
-ray-induced single-stranded DNA breaks, cell-free
extracts, and varying amounts of the 24-kDa fragment in the presence or
absence of NAD+. As shown in Fig.
3, only 7% of DNA breaks were rejoined
in the absence of NAD+ due to inhibition of DNA repair by
bound PARP. By contrast, when poly(ADP-ribosyl)ation and dissociation
of PARP from DNA breaks was initiated by addition of NAD+,
about 30% of DNA breaks were repaired. This NAD+-promoted
DNA repair was significantly inhibited by addition of the 24-kDa
fragment (Fig. 3, A and B). A 6.6-fold molar
excess of the 24-kDa fragment relative to PARP was sufficient to
inhibit NAD+-promoted DNA repair by 80% (Fig. 3,
A and B, 15 pmol of PARP derived from extract
versus 100 pmol of the 24-kDa fragment), consistent with the
hypothesis that the 24-kDa fragment, unlike full-length PARP, binds to
and persists on DNA breaks in the presence of NAD+.

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Fig. 3.
Cell-free DNA repair assay and reduced
synthesis of ADP-ribose polymers in the presence of the 24-kDa
fragment. A, cell-free reactions were carried out in
the presence or absence of NAD+ using varying amounts of
the 24-kDa fragment and damaged pBS (open circular, OC)
containing -ray-induced single-stranded DNA breaks in a 50-µl
reaction mixture (see "Materials and Methods"). The DNA was then
purified, and damaged pBS was separated from repaired closed circular
pBS by ethidium bromide, 1% agarose gel electrophoresis to determine
the amount of repaired plasmid. The relative proportions of the 24-kDa
fragment and PARP (15 pmol derived from cell-free extracts) are also
shown. B, repaired DNA (closed circular, CC)
expressed as a percentage of total DNA. C, cell-free DNA
repair reactions were carried out using varying amounts of the 24-kDa
fragment (0 pmol ( ), 50 pmol ( ), or 200 pmol ( )) and pBS
containing -ray-induced single-stranded DNA breaks in the presence
of 0.25 mM NAD+ and 1.3 µCi of
[32P]NAD+ in a 50-µl reaction mixture.
Reactions were also carried out using undamaged pBS ( ). The
32P-labeled protein was then trapped onto glass fiber
filters, and the radioactivity was counted to determine the amount of
ADP-ribose polymers synthesized during the reaction as described under
"Materials and Methods." The ratios between 0, 50, and 200 pmol of
the 24-kDa fragment to PARP (15 pmol derived from cell-free extracts)
were 0, 3.6 and 13.3, respectively.
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We then measured the amount of ADP-ribose polymers generated in the
cell-free assay in the absence or presence of the recombinant 24-kDa
fragment. As previously observed (28), incubation of cell-free extracts
with DNA breaks caused transient formation of ADP-ribose polymers (Fig.
3C). However, addition of the 24-kDa fragment significantly
inhibited ADP-ribose polymer formation, suggesting that the 24-kDa
fragment effectively competed with PARP in binding to DNA breaks and
thereby reduced the overall level of poly(ADP-ribosyl)ation in the
presence of NAD+.
Since ADP-ribose polymers are synthesized from NAD+,
inhibition of poly(ADP-ribose) formation should reduce the consumption of NAD+. To quantify the amount of NAD+ present
in the reaction mixture following the cell-free DNA repair assay,
[32P]NAD+ (32 nM) was added to
the assay and the reaction mixtures were applied to a 20%
polyacrylamide, 8 M urea gel (32) for quantification of
labeled NAD+. Activation of PARP consumed 20% of the
available NAD+, whereas addition of the 24-kDa fragment
(200 pmol) resulted in only a negligible reduction in NAD+
levels (data not shown).
Taken together, these results suggest that the 24-kDa fragment inhibits
DNA repair and ADP-ribose formation by binding to and persisting on DNA
breaks in competition with full-length PARP.
Binding of the 24-kDa Fragment to RNA Stem-Loop--
In nuclei,
PARP has been observed to associate with chromatin, particularly with
actively transcribed regions; this association has been shown to occur
by direct binding of PARP to transcribed RNA (14-16). We recently
demonstrated that binding of PARP to RNA stem-loop structures reduces
the rate of RNA elongation by RNA polymerase II and that formation of
DNA breaks and subsequent automodification of PARP removes the
transcriptionally inhibitory PARP molecules, thus up-regulating RNA
synthesis (17).
To investigate whether the 24-kDa fragment binds to RNA stem-loops,
uniformly 32P-labeled synthetic stem-loop RNA was prepared
by transcribing the TAR sequence from human immunodeficiency virus,
type I (see "Materials and Methods"), and was mixed with either the
24-kDa fragment or PARP. As shown in Fig.
4, PARP reduced the mobility of stem-loop
RNA on a native polyacrylamide gel, generating a discrete band. The
24-kDa fragment also reduced the mobility of TAR stem-loop RNA (Fig.
4), suggesting that the fragment, like full-length PARP, is capable of
binding to RNA stem-loops. The data in Fig. 4 reveal a linear
relationship between 32P activity associated with the
retarded fractions and the amount of 24-kDa fragment or PARP, enabling
us to conclude that the 24-kDa fragment has about 15% of the binding
activity of full-length PARP.

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Fig. 4.
Binding of the 24-kDa fragment and PARP to
RNA stem-loops. Recombinant PARP or the 24-kDa fragment was
incubated with 5 fmol of 32P-labeled TAR stem-loop RNA for
20 min at 37 °C in a 20-µl reaction mixture. The samples were
applied to native 6% polyacrylamide gels. The 32P activity
was visualized by autoradiography, and the 32P activity in
the retarded fraction was quantified with InstantImager (Packard);
relative activity is shown (arbitrary units).
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Suppression of Transcript Elongation by the 24-kDa
Fragment--
We then tested whether the 24-kDa fragment was capable
of reducing elongation of RNA transcripts using a reconstituted
pulse-chase elongation assay. Linearized p
Gf1, with a 90-base G-less
sequence at one end, was prepared (17) and used as a template. During the pulse, RNA polymerase II was loaded onto linearized p
Gf1 from
the end where the G-less sequence was located and allowed to transcribe
the G-less sequence in the absence of GTP but in the presence of ATP,
UTP, CTP, and [
-32P]CTP, resulting in the formation of
32P-labeled 90-base transcripts (Fig.
5A). The chase was initiated by adding GTP, excess unlabeled CTP, and either the 24-kDa fragment or
PARP. As shown in Fig. 5A, the formation of discrete labeled transcripts generated by pausing of RNA polymerase II at putative pause
sites was used to follow transcript elongation. Consistent with our
previous report showing that PARP suppresses transcript elongation
(17), addition of PARP inhibited the production of longer transcripts
(Fig. 5A). The addition of the 24-kDa fragment likewise
resulted in fewer of the longer transcripts (Fig. 5A), suggesting that the 24-kDa fragment also acted to suppress RNA synthesis by RNA polymerase II.

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Fig. 5.
Effect of the 24-kDa fragment on
transcription. A, pulse-chase transcription assay was
carried out with purified RNA polymerase II as described under
"Materials and Methods." During the pulse, RNA polymerase II was
loaded onto a G-less sequence located at one end of the linear template
in the presence of [ -32P]CTP, CTP, ATP, and UTP but
not GTP. RNA polymerase stalls at the first G, generating a 90-base
transcript. The chase was initiated by adding excess CTP and GTP in the
presence of the 24-kDa fragment (1 pmol) or PARP (0.1 pmol). Samples
were applied to a 6% acrylamide, 8 M urea gel, and
transcripts were visualized by autoradiography. B, a
cell-free run-off transcription assay was carried out for 1 h at
30 °C with HeLa nuclear extracts and the 24-kDa fragment in the
presence or absence of 0.25 mM NAD+ in a
10-µl reaction mixture. Transcripts were then fractionated on a 3%
polyacrylamide, 8 M urea gel, and the gel was exposed to
x-ray film to visualize the 400-base run-off transcripts. The relative
proportions of the 24-kDa fragment and PARP (2 pmol derived from HeLa
nuclear extracts) are also shown. C, produced run-off
transcripts expressed in arbitrary units.
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RNA stem-loop structures have been demonstrated to result in
pausing of RNA polymerase II during transcription (33). In our previous
report (17), we suggested that the reduced rate of transcript
elongation in the presence of PARP occurs as the result of PARP binding
to and stabilizing these stem-loop structures. Upon DNA damage,
automodification of PARP promotes the resolution of PARP-RNA complexes,
thereby up-regulating transcription (17). On the other hand, the 24-kDa
fragment lacks the automodification site and may therefore compete with
full-length PARP and inhibit its ability to up-regulate transcription
downstream of DNA damage. To test this hypothesis, we carried out
run-off transcription assays using HeLa nuclear extracts in the
presence or absence of the 24-kDa fragment. Reactions were carried out
using 1.8 transcription units of HeLa nuclear extract that contained
about 2 pmol of PARP. As shown in Fig. 5B, run-off
transcripts 400 bases long were produced from linearized pCMV-Luc. Upon
addition of NAD+, PARP that was bound to the ends of
linearized template DNA became automodified, and an increase in
transcripts was observed, consistent with our previous report (17).
However, the addition of the 24-kDa fragment to the run-off reactions
(i.e. in addition to PARP and NAD+) resulted in
the production of fewer transcripts (Fig. 5B). A 15-fold
molar excess of the 24-kDa fragment relative to PARP was sufficient to
reduce the NAD+-promoted transcription by 80% (Fig. 5,
B and C, 2 pmol of PARP derived from HeLa nuclear
extracts versus 30 pmol of the 24-kDa fragment), indicating
that these fragments in fact inhibit PARP-mediated up-regulation of RNA synthesis.
Increased Induction of Apoptosis in Cells Expressing the 24-kDa
Fragment--
To investigate whether expression of the 24-kDa fragment
biases cells toward apoptosis, HeLa S3 cells were transiently
transfected with pcDNA 3.1
/AF24 for 24 h. The transfected
cells were exposed to 50 µM MNNG for 20 min and then
incubated in nomal medium for an additional 2 h, after which
apoptosis was analyzed by TUNEL assay. Control cells were transfected
with pCMV-Luc for expression of luciferase. As shown in Fig.
6, B and F, only a
few TUNEL signals were detected in nontreated cells. On the other hand,
we found increased TUNEL signals following MNNG treatment of HeLa S3
cells transfected with pCMV-Luc (Fig. 6D, 39% positive
cells). Cells transfected with pcDNA 3.1
/AF24 and treated with
MNNG showed a further and dramatic increase in such TUNEL signals (Fig.
6H, 76% positive cells).

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Fig. 6.
Increased induction of apoptosis by
expression of the 24-kDa fragment. Plasmids containing cDNA
for the 24-kDa fragment (frames E-H) or control plasmids
containing luciferase cDNA (frames A-D) were
transfected into HeLa S3 cells. The cells were then incubated in
serum-free DMEM in the presence (C, D, G, and H)
or absence (A, B, E, and F) of 50 µM MNNG for 20 min, and apoptotic cells were analyzed by
TUNEL assay after 2 h of incubation in normal medium.
TUNEL-positive signals were then visualized by fluorescence microscopy
(B, D, F, and H). The number in each
frame represents the percentage of TUNEL-positive cells among 300 cells
analyzed. Phase contrast images are also shown (A, C, E, and
G).
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Quantitative Western blotting of transfected cells (using either the
C-II-10 monoclonal antibody, which recognizes the automodification domain of PARP, or the F1-23 antibody against zinc finger 2 of both
PARP and the 24-kDa fragment (26)) revealed a PARP concentration of 28 fmol per 1 × 104 cells versus 180 pmol of
the 24-kDa fragment per 1 × 104 cells. Since the
transfection efficiency was estimated to be greater than 90% (as
determined by the number of green fluorescent cells following
transfection with pcDNA 3.1
/GFP), we calculated that the ratio of
24-kDa fragment to PARP in HeLa S3 cells expressing the 24-kDa fragment
was 7:1. This ratio appears sufficient, therefore, to sensitize cells
to the apoptotic effects of MNNG.
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DISCUSSION |
During apoptosis, the abundant nuclear enzyme PARP is cleaved by
caspase-3, generating a 24-kDa fragment containing the DNA binding
domain (18-20). In this report, we have demonstrated that the 24-kDa
fragment, like full-length PARP, is capable of binding to both DNA
breaks and RNA, although the affinity of the 24-kDa fragment for DNA
breaks and RNA was reduced to 25 and 15% relative to full-length PARP,
respectively. However, given the high affinity of PARP for DNA breaks
(31) and RNA (34), the reduced affinity is still significant and
consistent with a functional role for the 24-kDa fragment in apoptosis.
Since apoptosis can be induced in PARP knockout cells (35-37),
cleavage of PARP may not be required for apoptosis per se.
However, it has been suggested that PARP functions to bias cells toward survival (21, 37, 38). If so, then a reduction in the level of
full-length PARP as a consequence of cleavage by caspase-3 may counter
this normal bias. In addition, the fact that the resulting 24-kDa
fragment retains the DNA binding domain of PARP but lacks the
automodification site may enable it to act as a dominant negative factor in relation to full-length PARP, since automodification regulates both the DNA binding activity and its poly(ADP-ribosyl)ation activity of PARP (5). In fact, we have demonstrated here that the
24-kDa fragment inhibited DNA repair, ADP-ribose polymer formation, and
damage-dependent up-regulation of transcription mediated by PARP, all of which could shift the cell bias from survival to death
through apoptosis.
Inhibition of DNA repair by the 24-kDa fragment can be accounted for by
the fact that the 24-kDa fragment, unlike full-length PARP molecules,
persists on DNA breaks, thus preventing DNA repair enzymes from gaining
access to sites of damage. DNA repair can similarly be inhibited under
conditions where PARP automodification is suppressed (6), since
automodification is essential for driving the dissociation of PARP from
DNA breaks. Alternatively, overexpression in mammalian cells of a
42-kDa PARP fragment containing the DNA binding domain and
automodification site but lacking the catalytic domain also inhibits
DNA repair due to persistence of the 42-kDa fragments on DNA breaks
(39). As expected, such inhibition increases the sensitivity of cells
to DNA-damaging agents (3, 39). Thus, inhibition of DNA repair by
binding of the 24-kDa fragment to DNA breaks may also sensitize cells
to DNA-damaging agents.
Binding of the 24-kDa fragment to DNA breaks reduced the
formation of ADP-ribose polymers (Fig. 3C) and consequently
the consumption of NAD+ (data not shown). It has been
suggested that depletion of NAD+ by activation of PARP
results in the reduced rate of ATP synthesis and causes cell death by
necrosis (23, 40-43). In fact, prevention of NAD+ and ATP
depletion stimulates apoptosis and suppresses induction of necrosis
(23, 41-43). Consistent with these observations, our biochemical
results also support the possibility that the 24-kDa fragment allows
cells to enter an apoptotic, rather than necrotic, pathway by
preventing over-consumption of NAD+.
The 24-kDa fragment caused inhibition of PARP-mediated up-regulation of
transcription (Fig. 5). We recently reported that PARP reduces the rate
of transcript elongation by RNA polymerase II and that activation and
automodification of PARP, as occurs in response to DNA damage, relieve
this inhibition, thereby resulting in up-regulation of transcription
(17). Since DNA-damaging agents induce RNA damage as well, we proposed
that such up-regulation allows cells to compensate for the collateral
loss of RNA following exposure to DNA-damaging agents, and that this
pathway is likely required for cell survival. The inhibition of such a
regulatory pathway by the 24-kDa PARP fragment may therefore sensitize
cells to DNA-damaging agents.
Following expression of the 24-kDa fragment in HeLa cells, Kim et
al. (43) found a 50% reduction in ADP-ribose polymer synthesis after exposure of the cells to UV light, as well as increased induction
of apoptosis. In the biochemical analysis reported here, a 50%
reduction in ADP-ribose polymer formation occurred in the presence of
about 120 pmol of the 24-kDa fragment (based on data in Fig.
3C) or about an 8-fold excess of 24-kDa fragment relative to
full-length PARP (15 pmol per reaction). In addition, consistent with
the report from Kim et al. (43), we also found increased induction of apoptosis by MNNG in HeLa cells expressing a 7-fold excess
of 24-kDa fragment relative to full-length PARP (Fig. 6). Furthermore,
our biochemical analysis shows that addition of a 7-fold excess of
24-kDa fragment relative to full-length PARP inhibited both
NAD+-promoted DNA repair (see Fig. 3B, 15 pmol
of full-length PARP derived from cell-free extracts versus
100 pmol of the 24-kDa fragment) and PARP-mediated up-regulation of
transcription (see Fig. 5C, 2 pmol of full-length PARP
derived from HeLa nuclear extracts versus 13 pmol of the
24-kDa fragment) by 80 and 60%, respectively. Thus, the 24-kDa
fragment, by competing with and acting in opposition to PARP, may
contribute to the process by which cells irreversibly commit to
apoptosis (44).