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INTRODUCTION |
Base excision repair
(BER)1 is one of the main
strategies of the cell for defense against exogenous and endogenous
genotoxic stress leading to single-base lesions in DNA (1, 2). Mouse embryonic fibroblasts (MEFs) rendered deficient in BER by gene deletion
of a BER enzyme are hypersensitive to monofunctional DNA-methylating
agents among other stressors, illustrating the importance of this
repair system (3-5). The current and generally accepted working model
for mammalian BER involves two sub-pathways, each proceeding as a
sequential process with several DNA and/or DNA-enzyme intermediates
(6-9). In each of the BER sub-pathways, repair can be initiated by
spontaneous base loss or DNA glycosylase action to produce an abasic
site and in some cases by coupled glycosylase base removal and strand
cleavage (1, 10, 11). When the intact abasic site is an intermediate,
DNA strand cleavage on the 5' side of the sugar is by the abundant
nuclear enzyme apurinic/apyrimidinic endonuclease (APE), and cleavage
on the 3' side of the sugar is by the deoxyribose phosphate (dRP) lyase activity of DNA polymerase
(
-pol) (12, 13). The single nucleotide gap is filled by
-pol, and then the nick is eventually sealed by a DNA ligase, thus completing the "short patch" or
"single nucleotide" BER sub-pathway (7, 14, 15). In mammalian
cells, repair of methylated bases, oxidized bases, and abasic sites
appears to occur predominantly by this sub-pathway (16, 17). In other cases, for example where the sugar of the abasic site is not removed efficiently, the "long patch" BER sub-pathway mediates repair (18).
This sub-pathway involves limited strand displacement and DNA synthesis
to replace between 2 and as many as 15 nucleotides in the damaged
strand (5, 19-21). Finally, this displaced damaged strand is excised
by the structure-specific flap endonuclease-1 (FEN-1), and the
resulting nick is sealed by a DNA ligase (14, 17, 20). The DNA
synthesis step of long patch BER can be conducted by
-pol or
other DNA polymerases (6, 18, 20, 22-26). The long patch BER
sub-pathway appears to account for only a small fraction of overall BER
in cell extracts that have proficient single nucleotide BER (23,
25).
The various steps in single nucleotide and long patch BER may be
coordinated via protein-protein and DNA-protein interactions, and
bimolecular complexes have been observed for x-ray cross-complementing factor 1 (XRCC1) and DNA ligase III (7, 27) and for
-pol and the
following: APE (28), PARP-1 (29), and DNA ligase I (14). Yet the
mechanism and regulation for each of these interactions are either
completely obscure or only beginning to be revealed, and the functional
implications of the interactions concerning the efficiency and accuracy
of BER are unknown. In the experiments to be described in this report,
we examined the interaction between PARP-1 and BER intermediates.
PARP-1 is an abundant nuclear enzyme that is known to bind to DNA nicks
and becomes activated to enzymatically poly(ADP-ribosyl)ate many
nuclear proteins, including itself, using NAD+ as
substrate. Self poly(ADP-ribosyl)ation of PARP-1 is known to cause it
to release from its DNA-binding site (reviewed in Ref. 30). PARP-1 in
mammalian cells exists in several isoforms (31-34). Recently, Dantzer
et al. (29) examined the role of PARP-1 in single nucleotide
and long patch BER using biochemical studies of "knock out" MEF
cell lines for the
-pol and/or PARP-1 genes. Among other
points, they found that extracts from PARP-1 null MEF were
moderately reduced in single nucleotide BER activity and strongly
reduced in long patch BER activity pointing to a role of PARP-1 in both
sub-pathways and a requirement for PARP-1 in long patch BER (29). A
role for PARP-1 in BER had been suggested much earlier, because of the
DNA binding specificity of PARP-1 for nicks in DNA (2, 35), and the
physical interactions of PARP-1 with the known BER proteins XRCC1 and
-pol (27, 29, 36). In addition, PARP-1 null MEF cells are known to
be hypersensitive to DNA-damaging agents that produce lesions repaired
by BER (37, 38). It was also found that PARP-1 can activate DNA ligase
during repair in vitro (39), and recently it was shown that
PARP-1,
-pol, and DNA ligase III/XRCC1 form a BER complex that can
support the ATP requirement for DNA ligation of the nick (40).
Therefore, it is considered well documented that PARP-1 has a role in
BER, yet the molecular mechanism of its participation has remained obscure.
In this study, we examined the question of whether proteins in MEF
crude extracts can be selectively labeled by a photoaffinity DNA probe
representing an early intermediate in long patch BER. We found that
only six proteins in the crude extract were strongly labeled by this
BER probe, including several well known BER enzymes. The results
described here reveal several important features of molecular
interactions between BER enzymes and BER intermediates.
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EXPERIMENTAL PROCEDURES |
Materials--
Rainbow colored molecular mass markers and
[
-32P]ATP were from Amersham Pharmacia Biotech.
Synthetic oligodeoxynucleotides were obtained from Oligos Etc.
(Wilsonville, OR) and from GENSET (La Jolla, CA). The photoreactive
dCTP analog,
exo-N-[
-(p-azidotetrafluorobenzamido)-ethyl]-deoxycytidine 5'-triphosphate (FAB-dCTP), was synthesized as described previously (41). Dulbecco's modified Eagle's medium (DMEM), GlutaMAX-1, L-glutamine, Hanks' balanced salt solution, and G418 were
from Life Technologies, Inc. Methyl methanesulfonate (MMS) and
3-aminobenzamide (3-AB) were from Sigma; 4-amino-1,8-naphthalimide
(4-AN) was from Acros Organics/Fisher, and cisplatin (Platinol-AQ) was
from Bristol-Myers Squibb Co. Fetal bovine serum (FBS) was obtained
from Summit Biotechnology (Ft. Collins, CO) and hygromycin from Roche
Molecular Biochemicals.
Cells and Extracts--
The wild-type MEF cell line
MB16tsA, clone 1B5, and the
-pol null cell line MB19tsA, clone 2B2,
have been described previously (4) and are available from ATCC (CRL
2307 and CRL 2308, respectively). These transformed fibroblast cell
lines were maintained in DMEM containing 10% FBS, GlutaMAX-1, and 80 µg/ml hygromycin in a 10% CO2 incubator at 34 °C. The
-pol null cells expressing a FLAG epitope-tagged
-pol were
prepared as described previously (42) and were maintained in DMEM, 10%
FBS, penicillin/streptomycin, GlutaMAX-1, and 600 µg/ml G418 in a
10% CO2 incubator at 34 °C. These cells were used here
in cross-linking experiments and were the MEF cell system used, unless
otherwise indicated. Wild-type and PARP-1 null spontaneously
immortalized MEFs (43) were obtained from Dr. Josianne
Ménissier-de Murcia (CNRS, Illkirch-Graffenstaden, France). The
cells were cultured at 37 °C in a 10% CO2 incubator in
DMEM containing L-glutamine and 10% FBS. All cells were
routinely tested and found to be free of mycoplasma contamination.
Extracts were prepared by suspending 5 × 106 cells in
100 µl of Buffer I (10 mM Tris-Cl, pH 7.8, 200 mM KCl) followed by addition of an equal volume of Buffer
II (10 mM Tris-Cl, pH 7.8, 200 mM KCl, 2 mM EDTA, 40% glycerol, 0.2% Nonidet P-40, 2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl
fluoride, 10 mg/ml aprotinin, 5 mg/ml leupeptin, 1 mg/ml pepstatin A).
The suspension was rotated at 4 °C for 1 h and centrifuged to
pellet cellular debris at 14,000 rpm for 10 min.
Cytotoxicity Assays--
Cytotoxicity was determined by growth
inhibition assays as described previously (25). Wild-type and
-pol
null cells or wild-type and PARP-1 null cells were seeded at a density
of 40,000 cells/well in 6-well dishes. The following day they were
exposed for 1 h to a range of concentrations of MMS or cisplatin
in growth medium. Both agents were added directly to the medium at the
time of the experiment. After 1 h, cells were washed with Hanks'
balanced salt solution, and fresh medium was added. Dishes were
incubated for 4-5 days until control untreated cells were ~80%
confluent. Cells (triplicate wells for each drug concentration) were
counted by a cell lysis procedure (44). Experiments were also conducted in the presence of PARP inhibitors 3-AB and 4-AN. Stock solutions of
both inhibitors were prepared in dimethyl sulfoxide and then diluted in
growth medium to the highest non-toxic concentration (10 mM
for 3-AB and 10 µM for 4-AN). Dilutions of MMS or
cisplatin were prepared in medium containing inhibitor, and the cells
were dosed as described above. Following the 1-h MMS or cisplatin
exposure, cells were washed and then incubated in PARP
inhibitor-containing medium for a further 23 h.
Proteins and Antibodies--
Human
-pol, FEN-1, and APE were
purified as described (18, 45, 46). Recombinant PARP-1 was purified by
conventional chromatographic methods employing anion and cation
exchange according to an adapted procedure (47). Human DNA polymerase
was provided by Dr. William C. Copeland (NIEHS, National Institutes
of Health). Human DNA ligase I and DNA ligase III were provided by Dr.
Alan E. Tomkinson (University of Texas Health Science Center, San
Antonio, TX). Human replication protein A (RPA) was isolated as
described (48). Monoclonal antibodies against PARP-1 were purchased
from PharMingen (San Diego, CA). Antibodies against DNA ligase I and RPA were purchased from NeoMarkers (Freemont, CA). Antibodies against
FEN-1 were provided by Dr. Vilhelm A. Bohr (NIA, National Institutes of
Health), and FLAG antibody-specific agarose was purchased from Sigma.
Primer-Template Used in the Cross-linking
Experiments--
Primer-templates used in the study are shown
in Table I.
Radioactive Labeling of Oligodeoxynucleotide
Primers--
Oligodeoxynucleotides were
5'-32P-phosphorylated with T4 polynucleotide kinase as
described (49). Unreacted [
-32P]ATP was separated by
passing the mixture over a Nensorb-20 column using the manufacturer's
suggested protocol.
Primer-Template Annealing--
Lyophilized oligodeoxynucleotides
were resuspended in 10 mM Tris-HCl, pH 7.4, and 1 mM EDTA, and the optical density was measured. Complementary oligodeoxynucleotides were annealed by heating a solution
of equimolar concentrations to 90 °C for 3 min, followed by slow
cooling to room temperature.
Photoaffinity Labeling--
The photoaffinity labeling reaction
mixture (10 or 20 µl) contained 50 mM Tris-HCl, pH 7.8, 10 mM MgCl2, 4 or 8 µl of 1.6 mg/ml cellular
extract (prepared from
-pol null MEF expressing a FLAG
epitope-tagged
-pol unless otherwise indicated), 0.4 µM 32P-labeled DNA containing either a
reduced abasic site (DNA1), a nicked abasic site
(DNA3), or a uracil residue (DNA8) (see Table I), and 20 µM FAB-dCTP. The reconstituted reaction
mixture (10 or 20 µl) contained 50 mM Tris-HCl, pH 7.8, 10 mM MgCl2, 50 mM KCl, 0.4 µM 32P-labeled DNA (DNA1), 20 µM FAB-dCTP, 0.2 µM PARP-1, 0.2 µM FEN-1, 1 µM
-pol, and 0.7 µM APE. The reaction mixtures were incubated at 25 °C
for 30 min to allow incorporation of the photoreactive probe
exo-N-[
-(p-azidotetrafluorobenzamido)-ethyl]deoxycytidine 5'-monophosphate (FAB-dCMP). Then the mixtures were spotted onto Parafilm, placed on ice, and irradiated with UV light
(
max = 312 nm, 5-7 mJ) with a UV Stratalinker
(Stratagene, La Jolla, CA). The photoaffinity labeled proteins were
separated by 10% SDS-PAGE. Gels were dried and subjected to autoradiography.
Introduction of Photoreactive FAB-dCMP Probe into DNA--
The
reaction conditions for evaluating DNA synthesis with photoreactive
FAB-dCTP or dCTP were identical to those used for the cross-linking
experiments. After the initial DNA synthesis for 30 min at 25 °C,
the reaction was terminated by adding 10 µl of 90% formamide, 50 mM EDTA, and 0.1% bromphenol blue. The mixture was heated
for 3 min at 80 °C, and products were analyzed by electrophoresis
followed by autoradiography.
Unlabeled DNA Competition for PARP-1 Labeling by Photoreactive
DNA--
PARP-1 binding to DNA5, DNA6, and
DNA7 (Table I) was evaluated for competition with PARP-1
labeling 32P-labeled photoreactive DNA3.
Unlabeled DNA5, DNA6, or DNA7 was used in a 1:1 molar ratio with the probe DNA. Reaction mixtures (10 µl) contained 50 mM Tris-HCl, pH 7.4, 10 mM
MgCl2, 4 µl of 1.6 mg/ml cellular extract, 0.4 µM 32P-labeled DNA3, 20 µM FAB-dCTP. Reconstituted system reaction mixtures (10 µl) contained 50 mM Tris-HCl, pH 7.8, 10 mM
MgCl2, 50 mM KCl, 0.4 µM
32P-labeled DNA3, 20 µM FAB-dCTP,
0.2 µM APE, and 0.2 µM
-pol. The
reaction mixture was incubated for 30 min at 25 °C with 0.4 µM of unlabeled competitor DNA, DNA5,
DNA6, or DNA7. The competitor DNA was added to
the reaction mixture before UV irradiation. For the reconstituted
system, 0.3 µM PARP-1, 0.2 µM APE, and 0.2 µM
-pol were used. UV light (
max = 312 nm, 3 mJ) irradiation was with a UV Stratalinker. Cross-linked proteins
were separated by 10% SDS-PAGE and visualized by autoradiography.
Immunoprecipitation Experiments with Antibodies against DNA
Repair Proteins--
Immunoprecipitation was as described (42) using
M2-FLAG antibody conjugated to agarose. Briefly, the photoaffinity
labeled reaction mixture (200 µl) was mixed with 5 µg of anti-FLAG
M2 antibody conjugated to agarose, incubated with rotation for 4 h
at 4 °C, and pelleted by centrifugation at 14,000 rpm (15 s). The
supernatant was discarded, and the protein-bound agarose complex was
washed 4 times with extract buffer. After a final wash, the buffer was
removed, and the protein agarose suspension was mixed with 50 µl of
SDS-gel loading buffer and incubated in a boiling water bath for 5 min
to release the antibody-bound proteins.
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RESULTS |
Effect of PARP Inhibitors on MMS-induced
Cytotoxicity--
To probe for enzymes recognizing BER intermediates,
we used crude extracts of paired MEF cell lines that were wild type or deleted in the genes for either
-pol or PARP-1. The respective wild-type cell lines appeared to be proficient in BER as revealed by
their resistance to MMS, a prototype agent for inducing DNA damage
repaired by BER (Fig. 1, A and
B). The
-pol null cell line exhibits hypersensitivity to
MMS (Fig. 1A), as is well known from previous work (4).
Cellular resistance to MMS in these fibroblasts also appeared to depend
on PARP-1, since the wild-type cell line was rendered extremely
hypersensitive to MMS in the presence of PARP inhibitors 3-AB or 4-AN
(Fig. 1A). The extreme hypersensitivity imposed by the PARP
inhibitors suggests that PARP is involved in both of the BER
sub-pathways, since our previous results indicated that each BER
sub-pathway results in greater resistance to MMS than that seen in the
presence of 4-AN (25). The PARP-1 null cells were also hypersensitive
to MMS (Fig. 1B), and with PARP inhibitors the wild-type
cells became even more sensitive than the PARP-1 null cells suggesting
a role in BER for other proteins with PARP activity in addition to
PARP-1. Therefore, in both of the MEF cell line systems used here, it
appears that PARP-1 plays a role in repair of MMS-induced lesions, and
we inferred that this reflects a role of PARP-1 in BER. As a control
for the cellular response to a "non-BER" genotoxicant, no such
hypersensitivity was observed when the
-pol null cell line was
exposed to cisplatin, and 4-AN did not render the wild-type cells
hypersensitive (Fig. 1C).

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Fig. 1.
Effect of PARP inhibitors on MMS- and
cisplatin-induced cytotoxicity. Survival curves were produced as
described under "Experimental Procedures." Wild-type (closed
symbols) and -pol null (open symbols) mouse
fibroblasts (A and C) or PARP-1 wild-type
(closed symbols) and null (open symbols) mouse
fibroblasts (B) were exposed for 1 h to MMS
(A and B) or cisplatin (C) in the
absence ( , ) or presence of 3-AB (10 mM for 24 h) ( ), or 4-AN (10 µM for 24 h) ( ). Data are
from representative experiments; values represent the mean of
triplicate determinations.
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Photoaffinity Labeling with a BER Intermediate--
The structure
of the photoaffinity labeling probe is illustrated in Fig.
2. The photoreactive dCTP analogue,
FAB-dCTP, and a 32P-5'-end-labeled 34-base pair
oligonucleotide containing a synthetic abasic site
(3-hydroxy-2-hydroxymethyltetrahydrofuran (THF) 5'-phosphate) (i.e. DNA1, Table
I), were used to produce the
photoreactive BER intermediate in situ (i.e.
DNA2, Table I). The arylazido moiety in the photoreactive
group (Fig. 2A) can be activated with near (312 nm)-UV light
and is therefore a more selective cross-linking group than those with
azido moieties requiring higher energy UV-light exposure such as 254 nm. The abasic site-containing DNA was cleaved by APE in the MEF
extract, and the photoreactive FAB-dCMP residue was introduced at the
resulting 3'-hydroxyl group by a DNA polymerase in the extract. As
illustrated in Fig. 3, this process
resulted in a photoreactive nick containing the THF abasic site sugar
phosphate at the 5'-margin and FAB-dCMP at the 3'-margin
(DNA2, Table I). Note that the sugar in the THF abasic site
was not subject to removal by a
-elimination mechanism.

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Fig. 2.
Structure of the photoreactive dCTP
analogue (FAB-dCTP) and oligonucleotide DNA (DNA1, Table
I). A, dC*TP,
(exo-N-[ -(p-azidotetrafluorobenzamido)-ethyl]deoxycytidine
5'-triphosphate), the photoreactive arylazido group with a spacer arm
was attached to the N-4 of dCTP. B, 34-base pair
oligodeoxynucleotide containing the tetrahydrofuran residue
(X), 3-hydroxy-2-hydroxymethyltetrahydrofuran
(THF), opposite G.
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Fig. 3.
Synthesis of the photoreactive DNA
probe. A, a scheme illustrating in situ
conversion of radiolabeled DNA1 (Table I) to the nicked DNA
containing FAB-dCMP and deoxyribose phosphate at the 3'- and
5'-margins, respectively. B, photograph of an autoradiogram
of a DNA sequencing gel showing primer extension products with FAB-dCMP
and dCMP by the MEF extract. DNA1 was incubated for 30 min
at 25 °C with extract and products were analyzed as described under
"Experimental Procedures." Lane 1, DNA1 was
preincubated with purified APE to produce labeled 15-mer primer;
lane 2, DNA1 with extract, but without FAB-dCTP;
lane 3, DNA1, with extract and FAB-dCTP.
C, incorporation of dCMP and FABdCMP in vitro
using purified APE and -pol, lanes 2 and 3,
respectively. Lane 1 shows the 32P-labeled
initial 15-mer primer, produced from DNA1 using purified
APE.
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Primer extension DNA synthesis leading to the formation of
photoreactive probe in the cellular extract is illustrated in Fig. 3B, and for reference, formation of the probe by a system
reconstituted with purified APE and
-pol is shown in Fig.
3C. The electrophoretic mobility of the primer extended with
FAB-dCMP is slower than that of the primer extended with dCMP (Fig. 3,
B and C). The resulting DNA probe is regarded as
a long patch BER intermediate because the 5'-margin sugar phosphate of
the nick is refractory to
-pol-mediated removal, and the persistence
of the sugar phosphate prevents ligation of the unrepaired BER
intermediate by DNA ligases. Limited strand displacement DNA synthesis
by
-pol followed by FEN-1 cleavage of the resulting flap structure
are key subsequent steps in the long patch BER sub-pathway. Yet
DNA2 is frozen at a stage prior to strand displacement,
because the 3' FAB-dCMP is a poor substrate for further primer
extension DNA synthesis.
Photoaffinity Labeling of BER Proteins in the MEF
Extract--
Incubation of the 32P-labeled DNA
photoaffinity labeling probe in a MEF crude extract, followed by
near-UV light exposure, resulted in labeling of several proteins. A
typical labeling pattern, when DNA2 is used as probe, is
shown in Fig. 4A, lane
2. Only six proteins in the crude extract were strongly labeled by
this probe, suggesting significant selectivity for this labeling
procedure. Three of the labeled proteins were in a molecular mass range
consistent with the well known BER enzymes APE,
-pol, and FEN-1, and
the abundantly labeled higher molecular mass product was consistent with PARP-1, DNA ligases, or DNA polymerases, among other proteins. Four of the six proteins were identified in the studies described below
and are labeled accordingly in Fig. 4A. For comparison, a
completely different cross-linking pattern was obtained by an alternative method involving UV light exposure at 254 nm and with DNA
of similar structure as DNA2, but without the FAB
photoaffinity labeling group (Fig. 4B). This result
illustrates the selectivity of the labeling procedure used here and of
the DNA2 probe.

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Fig. 4.
Photoaffinity labeling and identification of
photoaffinity labeled proteins in MEF cellular extract.
A shows a photograph depicting the pattern of cross-linking
of cellular proteins to photoreactive nicked DNA. Experiments were as
described under "Experimental Procedures." The
5'-32P-labeled DNA1 (0.4 µM) was
preincubated with the usual -pol system MEF cellular extract
expressing a FLAG epitope-tagged -pol for 30 min at 25 °C with 20 µM FAB-dCTP (lane 2) or without FAB-dCTP
(lane 1) to introduce the photoreactive FAB-dCMP moiety into
DNA1 (i.e. to form DNA2). Then the
mixtures were irradiated with UV light ( max = 312 nm).
The UV cross-linked products were separated by SDS-PAGE and visualized
by autoradiography. The positions of the identified BER proteins and
the protein markers are indicated. B shows the cross-linking
pattern of cellular proteins to DNA5 without the
photoreactive FAB-dCMP. 5'-32P-Labeled DNA5
(0.4 µM) was preincubated with MEF extract (as in
A) and irradiated with UV light ( max = 254 nm). The UV cross-linked products were separated by SDS-PAGE and
visualized by autoradiography. C shows the identification of
photoaffinity labeled products by supplementing known DNA repair
proteins to the extract. The reaction mixture containing
DNA1 and FAB-dCTP was incubated for 30 min at 25 °C and
then UV-irradiated either with no additional proteins (lane
1), 2 µM APE (lane 2), 1 µM
-pol (lane 3), or 3 µM FEN-1 (lane
4). D shows the identification of cross-linked proteins
in the MEF cellular extract by immunoprecipitation. After UV
cross-linking, as in A, the photoaffinity labeled proteins
were immunoprecipitated either with preimmune IgG (lane 2),
anti-FEN-1 antibody (lane 3), or anti-FLAG antibody
( -pol) (lane 4). Lane 1 represents 10% of the
cross-linked reaction mixture used for immunoprecipitation. The
positions of identified proteins and protein markers are indicated in
the right and left margins, respectively.
E shows a comparison of cross-linking patterns of cellular
proteins to photoreactive nicked DNA (DNA2) obtained with
-pol null cell extract (lane 3 and 4) and the
usual MEF cellular extract expressing a FLAG epitope-tagged -pol
(lanes 1 and 2). The 5'-32P-labeled
DNA1 (0.4 µM) was preincubated for 40 min at
25 °C with 20 µM FAB-dCTP (lanes 2 and
4) or without FAB-dCTP (lanes 1 and 3)
to introduce the photoreactive FAB-dCMP moiety into DNA. The level of
DNA synthesis for incorporation of FAB-dCMP was found to be similar for
the usual extract containing -pol and -pol null extract. The
mixtures were then UV light ( max = 312 nm)-irradiated,
and the UV-cross-linked products in the FEN-1 to APE molecular mass
range were visualized as in A.
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Several of the proteins strongly labeled in the experiment shown in
Fig. 4A, lane 2, were identified. We first
supplemented the MEF extract individually with well known BER enzymes
in purified form to determine if these proteins could be labeled and if
the products had gel mobility similar to those seen in Fig.
4A. With extract supplemented with APE,
-pol, and FEN-1
(Fig. 4C, compare lane 1 with lanes
2-4), an increase in labeling was observed for products migrating
at 42, 45, and 52 kDa, respectively. The mobility of each labeled
species was consistent with addition of one molecule of the labeled DNA
strand to APE,
-pol, and FEN-1, respectively. Also, with addition of
an excess of one enzyme, labeling of the other two enzymes was
diminished. The doublet of labeled
-pol seen in Fig. 4C,
lane 3, was consistent with the inherent mass difference
between the endogenous FLAG-tagged
-pol in the MEF extract and the
purified native
-pol added to the extract. Overall, these results
indicate that FEN-1,
-pol, and APE can be labeled by the probe when
added to the extract individually and that these purified proteins can
bind the probe and compete with other proteins in the extract.
We next examined the question of whether these three
labeled proteins in the MEF extract could be immunoprecipitated with antibody specific to each protein. Antibody to the FLAG epitope on
-pol precipitated labeled protein corresponding to the 45-kDa product (i.e. FLAG-
-pol) and a portion of the higher
molecular mass product labeled as PARP-1 in Fig. 4D,
lane 4. Antibody to FEN-1 precipitated labeled protein
corresponding to the 52-kDa product (Fig. 4D, lane
3). Our antibody to APE failed to precipitate any labeled protein
(data not shown), and similarly, non-immune IgG did not precipitate any
labeled proteins (lane 2). Since APE was not identified by
immunoprecipitation in these experiments, assignment of this protein as
the 42-kDa labeled product is considered only preliminary. Finally, the
proteins responsible for the two lower molecular mass labeled products
seen in Fig. 4A, lane 2 (designated I
and II), were not further evaluated, but they were not
precipitated by any of the antibodies noted above. The identity of
-pol was further confirmed using
-pol null cell extract instead of the usual MEF extract (Fig. 4E). The results of
cross-linking experiments indicated that the cross-linking product with
molecular mass consistent with
-pol was not detected with extract
from
-pol null cells (Fig. 4E, compare lane 2 to lane 4).
Next, we conducted experiments to identify the protein responsible for
the higher molecular mass product, which was the major labeling
product. The protein was eventually identified as PARP-1. In the first
experiment, purified PARP-1 was added to the extract (Fig.
5A). The amount of the higher
molecular mass product was observed to increase in proportion to the
amount of PARP-1 added to the extract, suggesting that this product was
due to PARP-1. This notion was further examined by adding
NAD+ to the reaction mixture. The premise of this
experiment is that in the presence of NAD+, PARP-1 will
become auto-poly(ADP-ribosyl)ated, and as a result, the gel mobility of
PARP-1 will change such that it will not enter the gel utilized in this
experiment. When our incubation mixture was supplemented with
NAD+ before the UV light cross-linking, the usual higher
molecular mass labeled product was not observed, and a significant
amount of labeled protein failed to enter the gel (Fig. 5B,
compare lanes 1 and 2). Labeling of FEN-1,
-pol, and APE was not altered by the addition of NAD+
(Fig. 5B, compare lanes 1 and 2).
Thus, the protein responsible for the higher molecular mass product
appeared to be poly(ADP-ribosyl)ated, but FEN-1,
-pol, and APE were
not. In the next experiment, we compared the amount of the higher
molecular mass product formed by cellular extracts prepared from either
wild-type or PARP-1 null MEF cell lines. This higher molecular mass
product was detected with the extract from the wild-type cell line but
not with extract from the PARP-1 null cell line (Fig.
5C).

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Fig. 5.
Identification of PARP-1. Several
approaches were used to identify the high molecular mass product.
A is a photograph of an autoradiogram showing only the high
molecular mass product (PARP-1). The reaction mixture containing
DNA1, FAB-dCTP, and cellular extract was preincubated for
30 min at 25 °C and then UV-irradiated ( max = 312 nm)
either with (lanes 2-4) or without (lane 1)
purified PARP-1. B shows the photocross-linking products
obtained in cellular extract in the absence (lane 1) or in
the presence (lane 2) of 1 mM NAD+
(lane 2). The positions of identified proteins and protein
markers are indicated. C shows the cross-linking of the high
molecular mass product in cellular extract derived either from
PARP-1( / ) (lane 1) or PARP-1(+/+) MEF (lane
2). D depicts a photograph of an autoradiogram of
protein fractions precipitated with preimmune IgG (lane 2),
anti-PARP-1 IgG (lane 3), or 10% of the reaction mixture by
SDS-PAGE. The positions of the identified proteins and protein markers
are indicated to the right and left of the
panel.
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Finally, we examined the question of whether the higher molecular mass
labeled product observed with the usual MEF extract could be
immunoprecipitated with antibody specific to PARP-1 (Fig. 5D). We found that this product was precipitated with the
-PARP-1 antibody but not with non-immune IgG, confirming that the
labeled protein was indeed PARP-1. FEN-1,
-pol, and APE were not
co-immunoprecipitated along with PARP-1 by the
-PARP-1 antibody. We
also examined whether purified PARP-1, FEN-1,
-pol, and APE could be
labeled using a mixture of these purified proteins. For an incubation
mixture containing all four proteins and DNA2 as probe, we
found four labeled products of masses identical to those seen with the
crude cell extracts (data not shown) corresponding to PARP-1, FEN-1,
-pol, and APE; similar labeling for
-pol and APE was found either with or without the other two enzymes (data not shown).
Taken together, these results demonstrate that the higher molecular
mass labeled product was due to PARP-1. Other DNA repair proteins, such
as DNA ligase I, DNA ligase III, RPA, and DNA polymerase
were also
tested individually for their ability to cross-link to the probe.
Although these purified proteins could be individually cross-linked,
the mobility of the labeled product in each case (data not shown) was
different than that of the major higher molecular mass labeled products
observed here, i.e. in Fig. 4A, lane
2, the signal designated as PARP-1. Furthermore, the high
molecular mass product did not immunoprecipitate with antibodies
specific for DNA ligase I, DNA ligase III, or RPA.
DNA Structural Requirements for PARP-1 Labeling--
To examine
further the specificity of PARP-1 labeling, we conducted competition
experiments using a 32P-labeled probe designated
DNA4 that was similar to the usual DNA probe described
above (DNA2) and three unlabeled competitor DNA (Table I
and Fig. 6). This DNA probe,
DNA4, lacked a phosphate group on the 5'-tetrahydrofuran
sugar in contrast to DNA2, yet we found that the amount of
PARP-1 labeling by these two probes, DNA4 and
DNA2, was similar. DNA4 was used as a labeled
probe in competition experiments because preparation of closely related competitor DNA was more straightforward. Cell extract containing the
probe was supplemented with an equimolar ratio of competitor DNA, and
the mixture then was irradiated with near-UV light. As shown in Fig. 6,
the amount of PARP-1 labeling was reduced by competitors
DNA5 and DNA6 (see Table I) but not by the
normal double-stranded DNA tested, DNA7. The results of the
competition experiments using a reconstituted system that included
purified PARP-1 were virtually identical (Fig. 6B). The
results indicate that the double-stranded DNA competitor,
DNA7, had much less affinity for PARP-1 than the probe DNA,
DNA4. In contrast, DNA5 with dCMP in the
position of the FAB-dCMP group in the probe, competed about 50% of the
labeling, suggesting that DNA5 and the probe had similar affinity for PARP-1. The competitor DNA with an A:C base pair in the
nick, DNA6, competed more strongly suggesting greater
PARP-1 affinity than that of DNA5 or the probe DNA.

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Fig. 6.
Competition of photoaffinity labeling of
PARP-1 with different DNAs. 32P-Labeled
DNA3 (0.4 µM) was preincubated in MEF
cellular extract (A) or in a reconstituted system
(B) with FAB-dCTP (20 µM) for 30 min at
25 °C and then 0.4 µM unlabeled DNA5
(A and B, lane 2), DNA6
(A and B, lane 3), or DNA7
(A and B, lane 4) was added before UV
irradiation. Lane 1 (A and B) shows
PARP-1 cross-linking in cellular extract (A) or in a
reconstituted system (B) without competitor DNA. Reaction
mixtures were irradiated with UV light ( max = 312 nm).
The UV cross-linked products were separated by SDS-PAGE and visualized
by autoradiography. The positions of identified proteins and protein
markers are indicated.
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Different Labeling Patterns with Single Nucleotide and Long Patch
BER Substrates--
Next, we made use of a modification in the
structure of the labeled probe to examine further the specificity of
PARP-1 labeling. We found that PARP-1 labeling was influenced by the
DNA structure at the 5'-margin of the nick. An alternate DNA structure
lacking a 5'-sugar moiety on the downstream polynucleotide
(DNA9, Table I and Fig. 7)
gave much less labeling of PARP-1 than DNA2 (Fig. 7A). In the case of DNA9, the probe contained
FAB-dCMP and phosphate at the 3'- and 5'-margins of a nick
(DNA9), respectively. Note that DNA9 was
processed from DNA8 in situ by the extract
(i.e. DNA9, Table I) but was not ligated into a
double-stranded DNA product (data not shown). APE was not labeled by
the DNA9 probe, yet
-pol was equally labeled by
DNA9 and DNA2 (Fig. 7A, lane 4). The photoreactive FAB-dCMP probe was inserted into
DNA1 and DNA8 with similar efficiency to give
rise to the extended products, DNA2 and DNA9,
respectively (data not shown). As a control for these experiments, we
found that similar results were obtained when the two probes were
prepared by an alternate procedure involving annealing three preformed
oligomers (Fig. 7B), instead of two. In this experiment,
initial DNA substrates were created by annealing the oligonucleotides
such that DNA3 contains 3'-OH and 5'-THF (without
5'-phosphate), and DNA10 contains 3'-OH and 5'-phosphate in
the margin of the nick, respectively (see Table I). These experiments
indicate that labeling of PARP-1 was much less when the DNA probe
lacked the 5'-abasic site sugar characteristic of the early BER
intermediate.

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Fig. 7.
Specificity of PARP-1 photocross-linking with
single nucleotide (S-N) or long patch
(LP) BER substrates. A,
32P-labeled DNA1 (lanes 1 and
2) or DNA8 (lanes 3 and 4)
were preincubated with MEF cellular extract for 30 min at 25 °C with
(lanes 2 and 4) and without (lanes 1 and 3) FAB-dCTP. Then the reaction mixtures were
UV-irradiated at 312 nm. Photocross-linked proteins were separated by
SDS-PAGE and visualized by autoradiography. B, lanes
1 and 2 and lanes 3 and
4 contain DNA3 and DNA10, as initial
DNA, respectively. DNA3 contains 3'-OH and 5'-THF, whereas
DNA10 bears 3'-OH and 5'-phosphate groups in the margin of
the gap. Photocross-linking and the product analysis are as in
A. Positions of identified proteins and protein markers are
indicated.
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Co-immunoprecipitation of BER Proteins with Anti-FLAG
Antibody--
We used the approach of immunoprecipitation with an
antibody to the FLAG epitope attached to
-pol to probe for protein
complexes in the MEF extract. The DNA2 probe/extract
mixture was irradiated, and the mixture was then subjected to
immunoprecipitation. The results revealed that a portion of the PARP-1
and FEN-1 in the extract were co-immunoprecipitated as labeled proteins
(Fig. 8, lane 3) but that this
was not the case for APE or other proteins. Negative controls for this
experiment failed to reveal any protein immunoprecipitation (Fig. 8,
lane 2), indicating that the result shown in Fig. 8,
lane 3, was specific for the PARP-1, FEN-1, and
-pol
complex. It should be noted that extensive washing of the protein/DNA/antibody-agarose mixture eventually releases FEN-1 from the
complex, as depicted in Fig. 4. This suggests that the interaction
between FEN-1 and the
-pol/PARP complex is weaker than the
interaction between
-pol and PARP.

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Fig. 8.
Immunoprecipitation of PARP-1, FEN-1,
and -pol cross-linked to
32P-labeled DNA2. Experiments were
conducted as described under "Experimental Procedures;" and as in
Fig. 4, A, an autoradiogram of the cross-linking pattern of
cellular proteins is shown. Lane 1, 10% of reaction mixture
used for immunoprecipitation; lane 2, protein fraction
precipitated with the preimmune IgG; lane 3,
protein fraction precipitated with anti-FLAG epitope antibody
(i.e. to -pol).
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DISCUSSION |
The identity of the various enzymes participating in mammalian BER
is a subject of active investigation. In this study, we used a
sensitive, novel photoaffinity labeling reagent to identify BER enzymes
in the crude extract of MEF cells. For the two BER sub-pathways, a
nicked DNA structure is considered a branch point in BER sub-pathway
choice, and one can consider a nicked DNA structure with a
reduced abasic site sugar, as used here, as representing an
early intermediate frozen at the stage just prior to dRP lyase action
in single nucleotide BER or at the stage just prior to strand
displacement DNA synthesis in the long patch BER sub-pathway (6, 18).
Thus, we evaluated cross-linking of MEF proteins to such a DNA
structure as a novel approach toward probing for cellular enzymes
involved in BER. The use of a 32P-labeled DNA with a
sensitive photoreactive group at the 3'-margin of the nick permitted
cross-linking with near-UV light instead of 254 nm UV light. This
photoreactive BER intermediate was synthesized in situ (most
likely by
-pol) using a base-substituted dCTP analog carrying a
photoreactive arylazido group at the fourth position of the base. The
dNTP analog is known to be a good substrate for DNA polymerases
(50-52) including
-pol (52). This property permits introduction of
the photoreactive dCMP moiety into the 3'-margin of a gap in DNA, thus
creating the photoreactive BER intermediate within the cell extract
(Fig. 3). The DNA probe does not participate in downstream processes of
BER in the cell extract, namely strand displacement DNA synthesis or
DNA ligation, because a polynucleotide ending in the FAB-dCMP moiety is
a very poor substrate for either process. We provide evidence here that
the main target for BER protein interaction with this DNA is the BER
intermediate structure, rather than the two ends of the DNA oligomer or
a nick with a 5'-phosphate group.
When irradiated with near-UV light, the DNA probe synthesized in
situ could be cross-linked to the DNA polymerase or, after its
dissociation, to other proteins capable of interacting with a nicked
DNA structure. We regard this latter possibility as likely, because an
excess of DNA probe over DNA polymerase or other individual cellular
proteins was used in these experiments. Therefore, cellular proteins
interacting preferentially with a photoreactive BER intermediate can be
selected from the proteins in the crude cellular extract. This approach
may have advantages in the study of specific transactions of DNA and
DNA enzymes in cellular extract systems.
We found that protein labeling with the cellular extract was highly
specific, as only six proteins were strongly labeled. Four of these
turned out to be well known members of the BER machinery, PARP-1,
FEN-1,
-pol, and APE. These proteins, except for APE, were
identified as the labeled products using various approaches including
immunoprecipitation with specific antibodies (Figs. 4 and 5). Two
additional proteins of molecular mass of ~30 kDa were observed to be
affinity labeled also, but their identity was not pursued further in
this study.
PARP-1 is an abundant cellular protein and is well known to be a nicked
DNA sensor (2, 30, 35). Therefore, it is not surprising that PARP-1 was
a target of labeling by the photoreactive BER intermediate used as
probe in these experiments. We found that PARP-1 was by far the most
heavily labeled protein and that several other well known BER proteins
such as DNA ligases I and III and XRCC1 were not labeled. In view of
the strong labeling of PARP-1 and the fact that PARP-1 has an
important, but as yet unknown role in BER, we chose to examine
requirements for PARP-1 labeling in detail. PARP-1 labeling in the MEF
extract used in these experiments satisfied several criteria of
affinity labeling using photoreactive nicked DNA; labeling varied as a
function of the structure of the DNA probe; labeling was highest for a nick carrying an abasic site sugar phosphate at the 5'-margin, indicating that PARP-1 recognition of this BER intermediate was sensitive and specific; labeling could be competed by oligonucleotides representing BER intermediates but not by double-stranded DNA.
Immunoprecipitation experiments with an anti-FLAG-
-pol antibody
system revealed some co-precipitation of PARP-1, FEN-1, and
-pol,
each cross-linked individually to a molecule of labeled DNA probe. This
indicated protein-protein interaction between PARP-1 and
-pol and is
in accord with the results reported by Dantzer et al. (29).
These workers found that PARP-1 interacts with the C-terminal portion
of
-pol in the presence of DNA (29). Furthermore, they used extracts
from cells made genetically deficient in
-pol and/or PARP-1 and
demonstrated that both proteins are involved in the BER of
uracil-derived abasic sites. In the absence of both PARP-1 and
-pol,
both BER sub-pathways were reduced (29). The deletion of PARP-1 had a
dramatic effect on long patch BER capacity measured in vitro
using the cell extract, yet the mechanism of the role of PARP-1 in BER
is still not clear (2, 30).
Our results on the requirements for PARP-1 recognition of BER
intermediates show that the enzyme interacts most avidly with DNA nicks
containing a sugar phosphate at the 5'-margin. This specificity of
PARP-1 toward binding a sugar moiety at the 5'-margin of a nick in a
single-stranded break suggests that PARP-1 recognizes a BER
intermediate with this structure. This intermediate is formed in BER
after introduction of a dNMP moiety by
-pol but before sub-pathway
choice leading to either single nucleotide BER or long patch BER.
Therefore, our data point to a potential role of PARP-1 in both BER
sub-pathways. The conclusion is consistent with the effect of PARP
inhibitors on MMS-induced cytotoxicity in the cell lines used in these
experiments. It appears that PARP plays a role in repair of MMS-induced
lesions, and we inferred that this reflects the role of PARP in both
BER pathways. We also found co-precipitation of PARP-1, FEN-1, and
-pol cross-linked to the DNA probe using antibodies to the FLAG
epitope attached to
-pol (Fig. 8). These data, again, show
interaction for these key BER proteins in a complex at a branch point
intermediate of BER. It is interesting that preferential PARP-1 binding
to this BER intermediate could stimulate the known rate-limiting steps of BER, such as the
-pol-dependent strand displacement
required in long patch BER and the dRP excision step required in single nucleotide BER. Thus, PARP-1 association in this way could be an
important event in the overall efficiency of BER and in sub-pathway selection. Work is currently in progress to explore these possibilities.