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J. Biol. Chem., Vol. 275, Issue 30, 23234-23239, July 28, 2000
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andFrom the Institut für Biochemie, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Federal Republic of Germany
Received for publication, March 22, 2000, and in revised form, May 4, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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In mammalian cells, the base excision repair
(BER) pathway is the main route to counteract the mutagenic effects of
DNA lesions. DNA nicks induce, among others, DNA polymerase activities
and the synthesis of poly(ADP-ribose). It is shown here that
poly(ADP-ribose) serves as an energy source for the final and
rate-limiting step of BER, DNA ligation. This conclusion was drawn from
experiments in which the fate of
[32P]poly(ADP-ribose) or [32P]NAD
added to HeLa nuclear extracts was systematically followed. ATP was
synthesized from poly(ADP-ribose) in a pathway that strictly depended
on nick-induced DNA synthesis. NAD was used for the synthesis of
poly(ADP-ribose), which, in turn, was converted to ATP by
pyrophosphorylytic cleavage utilizing the pyrophosphate generated from
dNTPs during DNA synthesis. The adenylyl moiety was then preferentially
used to adenylate DNA ligase III, from which it was transferred to the
5'-phosphoryl end of the nicked DNA. Finally, ligation to the 3'-OH end
resulted in the release of AMP. When using NAD, but not
poly(ADP-ribose), in the presence of 3-aminobenzamide, the entire
process was blocked, confirming poly(ADP-ribosyl)ation to be the
essential initial step. Thus, poly(ADP-ribose) polymerase-1, DNA
polymerase The maintenance of an intact genome is crucial to each individual.
Therefore, DNA damages need to be efficiently removed, which is
accomplished by complex DNA repair mechanisms (reviewed in Ref. 1). The
major pathway, BER,1 is
initiated by DNA glycosylases that cleave the base-deoxyribose glycosyl
bond of a damaged nucleotide residue. Then, endonucleases are recruited
that cleave the chain on the 5' side of the abasic site. As a result,
nicked DNA intermediates occur. Nicked DNA, in turn, triggers the
catalytic activities of DNA polymerase Several different enzymes with poly(ADP-ribosyl)ation activity (EC
2.4.2.30) have been described recently, but the major cellular pathway
of NAD catabolism in response to the appearance of DNA lesions has been
ascribed to the catalytic activity of the 116-kDa protein PARP-1 (Ref.
4; reviewed in Ref. 5). Besides a potential participation of PARP-1 in
transcription (6, 7), recombination, apoptosis, and necrosis (5), a
large number of molecular and genetic studies have clearly implicated PARP-1 activity in positively regulating BER (8, 9). Originally, it was
suggested that poly(ADP-ribosyl)ation may activate a DNA ligase
required for DNA repair in mammalian cells (10). Several further
investigations confirmed a positive influence of PARP-1 activity on DNA
repair, especially on DNA ligation (Refs. 11-13; reviewed in Ref. 9).
However, the actual function of PARP-1 in the BER process and the
mechanism whereby poly(ADP-ribose) synthesis stimulates ligation (14)
have still remained obscure (reviewed in Ref. 9). X-ray repair
cross-complementing protein-1 (XRCC1) was the first human gene product
isolated that mediates the cellular response to ionizing radiation
(15). This protein is apparently essential and required for the BER
pathway (16). Recent investigations demonstrated specific interactions
of XRCC1 with Pol Most situations requiring highly efficient DNA repair are accompanied
by a dramatic decrease of the cellular ATP concentration. Considering
that ligation represents the rate-limiting, ATP-dependent step in BER, it appears reasonable to expect a compensatory mechanism that would enable efficient DNA repair even in situations of energy deprivation. In the present report, it is demonstrated that
poly(ADP-ribose) synthesized by PARP-1 may serve as a source of ATP
that is specifically used for ligation. This finding provides a
molecular mechanism for previous observations, in vivo and
in vitro, demonstrating the specific stimulation of ligation
by poly(ADP-ribosyl)ation during BER.
In Vitro Assays--
All reactions were carried out with nuclear
HeLa cell extracts purchased from Promega or prepared (21). In standard
reactions (10 mM Hepes, pH 7.9, 10% glycerol, 7 mM MgCl2, 50 mM KCl, 100 mM NaCl, 0, 1 mM EDTA, 0.25 mM
dithiothreitol, 0.25 mM phenylmethylsulfonyl fluoride, 10 µg/ml aphidicolin), 8 µg of nuclear protein were incubated with 200 ng of nicked plasmid DNA. Further additions are indicated in the
legends to the figures. The reaction volume was 10 µl, and
incubations were conducted at 30 °C for the time periods indicated.
All data presented are representative of at least three independent
experiments. Nicked plasmid was obtained by controlled incubation of an
empty vector plasmid, pUC 9, with DNase I and subsequent purification
of nicked plasmids by CsCl ethidium bromide centrifugation.
Poly(ADP-ribose) was synthesized with 10 µg/ml purified recombinant
PARP-1 from 100 µM NAD as described before (22).
Synthesized polymers were freed of residual amounts of DNA by DNase
treatment followed by phenol/chloroform extraction and precipitation.
Thin Layer Chromatography--
Reactions were stopped by
precipitation with 10 volumes of acetone. Precipitated nucleotides were
redissolved in 10 mM Tris-HCl, pH 8.0, 0.1 mM
EDTA. Samples containing equal amounts of radioactivity (as estimated
by Cerenkov counting) were subjected to cellulose-coated plates
(Machery-Nagel). Chromatography was performed as described before (23)
using the solvent system isobutyric acid/25%
NH4OH/H2O (96/4/19 (v/v/v)). After separation
was completed, the cellulose plates were dried and subjected to autoradiography.
Electrophoresis--
Reactions were stopped, and proteins were
separated by 6% polyacrylamide gel electrophoresis. For analysis of
plasmids, reactions were stopped, and after phenol/chloroform
extraction, DNA was precipitated with ethanol, resuspended, and applied
to 1% agarose gels in 90 mM Tris, 90 mM boric
acid, 2 mM EDTA, pH 8.3, and 50 µg/ml chloroquine.
Treatment of HeLa Cells and Measurements of Endogenous Enzymatic
Activities--
HeLa cells were grown in suspension in RPMI 1640 medium supplemented with 10% fetal calf serum in a humidified 5%
CO2 atmosphere at 37 °C. A medium change of confluent
cells was performed 2 h before treatment with
methyl-N'-nitro-N'-nitrosoguanidine (MNNG). At a
density of 106 cells/ml, a 10-ml culture was treated with
100 µM MNNG for 5 min. The cells were then centrifuged
and washed twice, and the culture was continued in a culture flask at
37 °C. 1-ml aliquots of the cell culture were taken after the time
intervals indicated, and nuclear extracts were prepared (21).
Poly(ADP-ribosyl)ation and nick-induced DNA synthesis activities of
nuclear extracts were determined under the same conditions as described
above. However, the 10-µl reactions contained only 0.5 µg of
nuclear protein. Incorporation of ADP-ribose was measured by using 1 µM [ First, the metabolism of 
, and ligase III interact with x-ray repair
cross-complementing protein-1 within the BER complex, which ensures
that ATP is generated and specifically used for DNA ligation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Pol ) and
poly(ADP-ribose) polymerase-1 (PARP-1) (2-4).
, DNA ligase III (Lig III), and PARP-1 (13,
17-20). Therefore, the complex of these proteins is supposed to
control BER (1, 9, 19).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]NAD (NEN Life Science Products)
and incubating for 15 min at ambient temperature. Incubations were
stopped by trichloroacetic acid precipitation. Incorporation of
32P-labeled ADP-ribose into washed precipitates was
determined by Cerenkov counting. Nick-induced DNA synthesis was
measured by using 0.1 mM dATP, dGTP, and dTTP and 1 nM [-32P]dCTP. Incubations were continued
for 30 min at 30 °C. The reactions were stopped, and after
phenol/chloroform extraction, DNA was precipitated with ethanol,
resuspended, and subjected to agarose gel electrophoresis. The gel was
dried and subjected to autoradiography. Incorporation of labeled
nucleotides was determined by Cerenkov counting of excised gel pieces.
All data presented are representative of at least three independent experiments.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P-labeled deproteinized
poly(ADP-ribose) (PAR) incubated in the presence of nicked DNA was
analyzed in HeLa nuclear extracts. The major product of PAR degradation is ADP-ribose (Fig. 1A, lane
1) arising from the activity of poly(ADP-ribose) glycohydrolase
activity present in nuclear extracts (24, 25). In the presence of 1 mM deoxynucleotides (dNTPs), which allowed nick-induced DNA
synthesis, in addition to radiolabeled ADP-ribose and AMP, labeled ATP
was detected (Fig. 1A, lane 2). Addition of only one or two
unlabeled dNTPs failed to support the generation of labeled ATP. A
mixture of at least three dNTPs enabled the synthesis of labeled ATP
from labeled PAR (not shown). On the other hand, addition of 1 mM sodium pyrophosphate instead of dNTPs did not give rise
to the generation of significant amounts of ATP (Fig. 1A, lane
3). Thus, synthesis of ATP strictly depended on DNA synthesis.
Moreover, the requirement for dNTPs indicated that the pyrophosphate
released during DNA synthesis is specifically channeled to the
ATP-forming activity, because added pyrophosphate was inefficient. It
should be pointed out that the only feasible direct pathway for the
generation of ATP from poly(ADP-ribose) includes the cleavage of the
phosphodiester bond of ADP-ribose by a pyrophosphorylase, yielding ATP
and ribose-5'-phosphate (26). If radioactively labeled ADP-ribose was
used in similar reactions, synthesis of ATP was only detected at very
high concentrations (10 mM) of pyrophosphate (not
shown).
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Fig. 1.
Poly(ADP-ribose) as specific source of ATP
used for ligation during DNA repair. A-C, thin layer
chromatographic analyses (autoradiograms) of nucleotides following
incubations of HeLa nuclear extracts in the presence of nicked DNA (see
"Experimental Procedures") and the compounds indicated below.
a, incubations contained 10 µM
-32P-labeled PAR and were performed for 20 min. The
sample in lane 2 contained, in addition, 300 µM dNTPs, whereas for lane 3, 1 mM
sodium pyrophosphate was added. b, incubations with 300 µM dNTPs and 10 µM
[-32P]NAD (lanes 1-5) or
[-32P]PAR (lanes 6-8) were performed for
the time intervals indicated. The samples used for lanes 5 and 10 contained, in addition, 50 µM 3-AB and
were incubated for 20 min. c, incubations were conducted
with 10 µM [-32P]ATP for the time
indicated. d and e, modification of HeLa nuclear
proteins by [-32P]NAD or [-32P]ATP
(autoradiograms of SDS-PAGE) during incubations in the presence of 300 µM dNTPs and the compounds indicated below. D,
incubations of nuclear HeLa extracts in the presence of 100 nM [-32P]NAD were continued for the times
indicated and then immediately subjected to electrophoresis, except for
the sample represented by lane 4. After 20 min of
incubation, this sample was treated with 40% trichloroacetic acid for
1 h at 37 °C, and the precipitated proteins were then subjected
to SDS-PAGE. The asterisks indicate fragments of PARP-1 as
detected by Western blot analysis using anti-PARP-1 antibodies.
E, nuclear HeLa extracts were preincubated for 10 min with
300 µM dNTPs in the absence (left lane) or
presence (right lane) of 1 mM unlabeled NAD.
Thereafter, 1 nM [-32P]ATP was supplied,
and incubation was continued for 20 min. F, nicked plasmid
was incubated for 20 min with nuclear HeLa extract,
[-32P]NAD, and a mixture of dGTP, dTTP, ddATP, and
ddCTP (500 µM each) in the absence (left lane)
or presence (right lane) of 50 µM 3-AB. After
extraction and precipitation, DNA was separated in an 1% agarose gel
and 50 µg/ml chloroquine, which was then dried and subjected to
autoradiography.
The dependence of ATP generation on the presence of ADP-ribose polymers
was further supported by incubations of nuclear extracts with dNTPs and
-32P-labeled NAD (Fig. 1B, lanes 2-4). The
synthesis of labeled ATP from labeled NAD was not detected, if the
reaction was performed in the presence of the inhibitor of
poly(ADP-ribosyl)ation, 3-aminobenzamide (3-AB) (Fig. 1B, lane
5). However, the presence of 3-AB did not prevent the synthesis of
radiolabeled ATP from labeled PAR (Fig. 1B, lane 10).
Consequently, dNTP-dependent formation of ATP exhibited an
absolute requirement for PAR, either added directly or synthesized by
endogenous PARP activity. ATP generation from labeled NAD or labeled
PAR (Fig. 1B) attained a maximum after about 20 min of incubation (compare in Fig. 1B, lanes 3 and 8 versus
lanes 4 and 9). If an amount of
[-32P]ATP equivalent to that of NAD or ADP-ribose
units in PAR was used instead, it was almost entirely metabolized to
ADP. Also, in this case, only a very little AMP (<1%) was formed
(Fig. 1C, right lane). In contrast, ATP synthesized from PAR
coupled to DNA synthesis was apparently not degraded to ADP (see Fig.
1, A and C). This suggests that the PAR-derived
ATP is inaccessible to ATPases, such as topoisomerases, helicases, or
kinases, but is accessible only to enzymes metabolizing ATP to AMP.
Thus, the pathway of ATP synthesis using PAR as intermediate product
appeared to provide ATP for a specific reaction.
In the first step of DNA ligation, ATP is used to form an adenylated
ligase intermediate. In subsequent steps, the adenylyl group is
transferred to the 5'-phosphoryl donor, and eventually, AMP is released
when the 5'-phosphoryl and the 3'-OH ends are joined. Owing to the fact
that both PARP-1 and Lig III are constituents of the BER complex
(17-20), it appeared to be a likely possibility that Lig III
(molecular weight, 103,000) is specifically using ATP generated from
PAR as described above. To test this possibility, nuclear extracts were
incubated in the presence of nicked DNA, dNTPs, and
-32P-labeled NAD for several time intervals, similarly
to reactions shown in Fig. 1B, lanes 2-4. Proteins were
then separated by SDS-PAGE and subsequently subjected to
autoradiography (Fig. 1D). As expected, the major
radioactively labeled protein represented automodified endogenous
PARP-1 (molecular weight, 116,000) or PARP-1-fragments (Fig. 1D,
asterisks), in accordance with previous reports showing that the
predominant reaction of poly(ADP-ribosyl)ation represents automodification of PARP-1 itself (27). A further radiolabeled protein
with an apparent molecular weight of about 100,000 was detected (Fig.
1D, arrow on right), the modification of which was sensitive
to treatment with acid (Fig. 1D, lane 4, arrow on right).
Compared with poly(ADP-ribosylation), adenylation of proteins is
relatively unstable, especially at acidic pH. Incubation of nuclear
extracts with [-32P]ATP and separation of proteins on
SDS-PAGE followed by autoradiography visualized proteins modified by
adenylation (Fig. 1E, left lane). As expected, several
adenylated proteins exhibited apparent molecular masses corresponding
to those of known human DNA ligases (28), i.e. Lig I (125 kDa), Lig III (103 kDa), and Lig II (70 kDa) (Fig. 1E, left
lane). If, prior to the adenylation reaction using
[-32P]ATP, unlabeled NAD and dNTPs were incubated with
the nuclear extract, the pattern of modified proteins was similar,
except that the 103-kDa protein (most likely Lig III) was not labeled. Obviously, this protein had already been adenylated by unlabeled ATP
formed during the preincubation in the presence of NAD and dNTPs.
Modification of Lig III directly from dNTPs or NAD was excluded by
adding an inhibitor of poly(ADP-ribosyl)ation: if the preincubation
with NAD and dNTPs was conducted in the presence of 3-AB, labeling of
the 103-kDa protein with [-32P]ATP was undiminished
(not shown).
The subsequent step after adenylation of DNA ligases is the activation
of the donor DNA by transferring the adenylyl group to the
phosphorylated 5'-end of a DNA nick. The resulting DNA-AMP complex then
reacts with the 3'-OH acceptor group, leading to the ligation of the
phosphorylated 5'-end with the 3'-OH under release of AMP. The
intermediate DNA-AMP complex can be trapped, if the final joining step
of the ligation is prevented. This was accomplished by using
2'-3'-dideoxy NTPs. The specific synthesis of ATP from PAR (see Fig. 1)
was still significant, if a mixture of ddATP, ddCTP, dGTP, and dTTP was
used instead of dNTPs (not shown). Therefore, during nick-induced DNA
synthesis, the incorporation of ddAMP or ddCMP into nicked plasmids
would lead, in part, to DNA-AMP intermediates of nicked plasmids with
2'-3'-dideoxy-ends adjacent to an adenylated 5'-end. This possibility
was verified in ligation reactions in nuclear extracts using nicked
DNA, -32P-labeled NAD, and unlabeled ddATP, ddCTP, dGTP,
and dTTP (Fig. 1F). The formation of labeled
DNA-[32P]AMP complexes was clearly detectable, but only
in the absence of 3-AB (Fig. 1F), that is, only if
poly([-32P]ADP-ribosyl)ation was allowed to occur.
These observations provide direct evidence for the conclusion that the
adenylyl moieties of poly(ADP-ribose) may be used to activate the
5'-phosphoryl ends of nicked DNA. In this NAD-dependent
pathway, the activities of poly(ADP-ribosyl)ation, DNA synthesis, and
ligation contribute directly to the ligation of nicked DNA only when
combined together.
All the enzymes implicated in this mechanism (PARP-1, Pol , and Lig
III) interact with the scaffold protein XRCC1 within the BER complex.
Heterodimerization of XRCC1 and Lig III leads to enhanced ligation
activity (20). On the other hand, interaction of XRCC1 with either
PARP-1 or Pol results in a down-regulation of the respective
catalytic activity (13, 18). It was confirmed in this study that in
cultured HeLa cells during BER Pol and PARP-1 activities are indeed
similarly regulated (Fig. 2A).
Treatment of HeLa cells with 100 µM MNNG, a well known
inductor of the BER pathway, led to transient activation of both PARP-1
and Pol activities followed by a sharp decline after about 30 min.
The assay of DNA synthesis used in this study was restricted to Pol activity, because other known DNA polymerases were inhibited by
aphidicolin (29). As tested for PARP-1 in quantitative Western blot
analyses (not shown), the modulation of activity was not caused by
changes of protein expression. This holds true also for the
reactivation after about 3 h following the MNNG treatment. These
in vivo experiments support the conclusion of previous
studies that during DNA repair both PARP-1 and Pol activities are
regulated in concert by their interaction with XRCC1. The time course
of the activation of PARP-1 and Pol also paralleled the enhanced occurrence of poly(ADP-ribose) and depletion of the cellular NAD, ATP,
and dNTP pools reported in earlier studies (30, 31). The onset of the
final step of BER, ligation, coincides with these events. The ligation
step is most important for successful DNA repair; it is rate-limiting
and ATP-dependent.
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The data presented so far suggested that synthesis of poly(ADP-ribose)
may serve as an emergency device to complete DNA repair under
conditions of cellular ATP depletion. It would be expected then that
availability of sufficient ATP should suppress such a pathway. This
hypothesis was tested by adding 5 mM ATP to incubations of
HeLa nuclear extracts in the presence of nicked DNA, dNTPs, and
-32P-labeled NAD. Surprisingly, under these conditions,
modification of nuclear proteins other than PARP-1 or its fragments was
not just inhibited but virtually absent (Fig. 2B, left panel,
lane 2), as opposed to the poly(ADP-ribosyl)ation observed in the
absence of ATP (lane 1, cf. also Fig. 1D).
Moreover, 32P adenylation of Lig III was also not
detectable. Analysis of radiolabeled nucleotides confirmed that in the
presence of 5 mM ATP, hardly any of the added
[-32P]NAD was metabolized (Fig. 2B, right panel,
lane 2). Addition of other NTPs, dNTPs, ADP, or AMP at the same
concentration (5 mM) did not cause any comparable effect. A
direct effect of ATP on PARP-1 was also excluded, because the catalytic
activity of the isolated enzyme did not exhibit such a sensitivity
toward ATP (not shown). Moreover, addition of isolated recombinant
automodification domain of PARP-1 (amino acids 337-573) resulted in
the partial recovery of poly(ADP-ribosyl)ation in the presence of ATP
(Fig. 2B, lanes 3). However, adenylation of Lig III was
still not detectable. The automodification domain of PARP-1 is known to
mediate specific interaction with partner proteins (22), in particular
XRCC1 (18). Therefore, it appears that excess of this domain affected the binding of the endogenous PARP-1 to XRCC1, and thus to the BER
complex. Consequently, PARP-1 inhibition was abolished, but the BER
complex was disabled to synthesize ATP.
The suggested effect of the added automodification domain of PARP-1 was
further supported by analysis of nick-induced DNA synthesis and
ligation catalyzed by the nuclear extracts. Whereas DNA synthesis, but
no ligation, was observed in the absence of ATP (Fig. 2C, lanes
1), the presence of 5 mM ATP led to ligation but
suppressed nick-induced DNA synthesis (Fig. 2C, lanes 2). Addition of the automodification domain of PARP-1 restored nick-induced DNA synthesis in the presence of ATP. Still, the ligated plasmid (closed circle) did not contain radiolabel of the added dNTPs (Fig.
2C, lanes 3). It is well known that DNA repair processes need a high level of ATP. Therefore, in vitro assays are
usually conducted in the presence of about 2 mM ATP and a
regenerating system (32). It was demonstrated in the experiment shown
in Fig. 2D that in the absence of any added ATP or an ATP
regenerating system nicked plasmids can be rejoined by HeLa nuclear
extracts. However, rejoining occurs only if the conditions established
above for the ATP synthesis from PAR are met. That is, conversion of nicked circles to closed circles was only observed if PAR (either directly added or formed from NAD by endogenous PARP-1) and dNTPs were
present (Fig. 2D, lanes 5 and 6). As mentioned
before, added ADP-ribose did not serve as a substitute for PAR.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The results of the present study support the conclusion that
poly(ADP-ribosyl)ation may directly contribute to the process of BER by
providing a source of ATP for the ligation step. It is important to
note that the favorable effects of NAD and PARP on ligation have long
been known. Originally, the observed stimulation of strand break
rejoining was suspected to be caused by poly(ADP-ribosyl)ation of a
ligase (10). However, this suggestion has not been confirmed. In
initial in vivo studies, antisense RNA expression was used to deplete cells of PARP-1. It was observed that the absence of PARP-1
resulted in a significant delay of DNA strand break rejoining (11).
Moreover, analyses of cells derived from PARP-1-/- mice
also revealed a substantial reduction of the DNA ligation activity
(12). In the meantime, the BER complex has been well studied, and
direct interactions of the proteins involved have been established,
both in vitro and in vivo. It was clearly
demonstrated that NAD, which provides the substrate for
poly(ADP-ribosyl)ation, accelerates the ligation step without
influencing, for example, Pol activity (13).
Considering the dramatic decrease of the cellular ATP concentration
following exposure to genotoxic agents (30, 31), a role of
poly(ADP-ribosyl)ation may be the extraction of energy from NAD and its
immediate use for DNA repair, specifically ligation. Along this line,
the high affinity of PARP-1 for DNA nicks would ensure that NAD is
preferentially used at sites in need of DNA repair. Taking into account
previous reports proposing the existence of a functional complex
consisting of XRCC1, DNA polymerase , Lig III, and PARP-1 (see Ref.
9 for review), the capability of these constituents of the BER complex
to produce ATP from poly(ADP-ribose) provides a rationale for the tight
interaction of these proteins (Fig. 3).
In addition, the stimulatory influence of NAD specifically on the
ligation reaction can be attributed to the generation of ATP, which is
apparently directly channeled to the ligase.
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According to the data presented, the pathway of ATP synthesis does not
necessarily include the cleavage of poly(ADP-ribose) to ADP-ribose.
However, ADP-ribose may be generated within the tight complex of
participating enzymes and may not be substituted for by added
ADP-ribose, similar to the situation found for pyrophosphate (Fig.
1A, lane 3). ATP formation using pyrophosphate generated from dNTPs during nick-induced DNA synthesis is difficult to
demonstrate directly, because the 
-phosphate group of only a single
added [-32P]dNTP is readily detectable in ATP (not
shown), presumably owing to transphosphorylation of ADP in the nuclear
extracts. Nevertheless, the requirement of DNA synthesis to generate
ATP from PAR strongly suggests the involvement of pyrophosphate.
Furthermore, high concentrations (10 mM) of added
pyrophosphate were effective to synthesize ATP from PAR in the absence
of dNTPs (not shown). Therefore, it would appear that the detected ATP
synthesis includes pyrophosphorolytic cleavage of ADP-ribose, yielding
ribose phosphate as its second product. An enzymatic activity
catalyzing this kind of reaction has been detected previously in HeLa
cell extracts (26).
It is of importance to the emerging model (Fig. 3) that the specific
generation of ATP requires the synthesis of DNA, which is accompanied
by the liberation of pyrophosphate. It is this ATP that is at least
preferentially used by Lig III for its autoadenylation and subsequent
activation of the DNA donor (5'-phosphorylated end) and, thus, mediates
ligation. Moreover, the final phase of BER, including nick-induced DNA
synthesis, generation of ATP from PAR, and ligation, appears to be
accomplished by an autonomous complex. Bulk phase ATP and pyrophosphate
are apparently not used by this complex. Rather, a high cellular energy
state (that is, high ATP concentrations) may possibly serve as a signal
that favors direct ligation of nicked DNA or execution of the Pol
-dependent short-patch repair pathway (2, 33).
An important observation (Fig. 2B) relates to the potential
function of Lig III and PARP-1 as molecular nick sensors. Both proteins
contain highly similar zinc-finger motifs that exhibit high affinity to
single strand breaks (34). According to the data presented in Fig. 2,
B and C, at high ATP concentrations, Lig III is
readily adenylated. At the same time, poly(ADP-ribosyl)ation and DNA
synthesis are strongly inhibited. A possible explanation would be that
adenylation of Lig III within the BER complex enhances its affinity to
DNA nicks or at least causes the BER complex to assume a conformation
that prevents DNA binding of PARP-1 and thereby poly(ADP-ribosyl)ation.
Such a mechanism would restrict the use of NAD as an energy source to
emergency situations of ATP shortage.
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ACKNOWLEDGEMENT |
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We thank G. Buchlow for technical assistance.
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FOOTNOTES |
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* This research was supported by the Deutsche Forschungsgemeinschaft.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.
To whom correspondence should be addressed. Tel.: 49-30-838-52910;
Fax: 49-30-838-56509; E-mail: lity@zedat.fu-berlin.de.
Published, JBC Papers in Press, May 9, 2000, DOI 10.1074/jbc.M002429200
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ABBREVIATIONS |
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The abbreviations used are:
BER, base excision
repair;
3-AB, 3-amionobenzamide;
Lig, DNA ligase;
MNNG, methyl-N'-nitro-N'-nitrosoguanidine;
PAGE, polyacrylamide gel electrophoresis;
PAR, poly(ADP-ribose);
PARP-1, poly(ADP-ribosyl) polymerase-1;
Pol , DNA polymerase ;
XRCC1, x-ray repair cross-complementing protein-1.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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) and nick-induced DNA synthesis (×) activities were determined
(see "Experimental Procedures") and related to the values obtained
for untreated cells (100%). B, nuclear HeLa extracts were
incubated with nicked DNA, 300 µM dNTPs, and 100 nM [