From the UPR 9003 du CNRS, Ecole Supérieure de
Biotechnologie de Strasbourg, Boulevard Sébastien Brant,
F-67400 Illkirch, France, the
Institut Curie Recherche UMR 147, Rue d'Ulm, F-75248 Paris Cedex, France,
Pharmaceuticals Research, BASF AG, D-67056
Ludwigshafen, Germany, and the ** Institut de Biologie
Moléculaire et Cellulaire, UPR du 9021 CNRS, 15 rue René
Descartes, F-67000 Strasbourg, France
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ABSTRACT |
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Poly(ADP-ribosylation) is a post-translational
modification of nuclear proteins in response to DNA damage that
activates the base excision repair machinery. Poly(ADP-ribose)
polymerase which we will now call PARP-1, has been the only known
enzyme of this type for over 30 years. Here, we describe a cDNA
encoding a 62-kDa protein that shares considerable homology with the
catalytic domain of PARP-1 and also contains a basic DNA-binding
domain. We propose to call this enzyme poly(ADP-ribose) polymerase 2 (PARP-2). The PARP-2 gene maps to chromosome 14C1 and
14q11.2 in mouse and human, respectively. Purified recombinant mouse
PARP-2 is a damaged DNA-binding protein in vitro and
catalyzes the formation of poly(ADP-ribose) polymers in a
DNA-dependent manner. PARP-2 displays automodification properties similar to PARP-1. The protein is localized in the nucleus
in vivo and may account for the residual poly(ADP-ribose) synthesis observed in PARP-1-deficient cells, treated with alkylating agents or hydrogen peroxide.
In response to DNA-strand breaks introduced either directly by
ionizing radiation or indirectly following enzymatic incision at a DNA
lesion, the immediate poly(ADP-ribosylation) of nuclear proteins
converts the DNA ends into intracellular signals that modulate DNA
repair and cell survival programs. At the sites of DNA breakage,
poly(ADP-ribose) polymerase
(PARP)1 (EC 2.4.2.30)
catalyzes the transfer of the ADP-ribose moiety from its substrate
NAD+, to a limited number of proteins involved in chromatin
architecture, DNA repair, or in DNA metabolism including PARP itself
(1-4). Recently, the generation of PARP-deficient mice by homologous recombination (5, 6) has clearly demonstrated the involvement of PARP
in the maintenance of the genomic integrity due to its role during base
excision repair (7-9). An substantial delay in DNA strand-break repair
was observed following treatment of PARP-deficient cells with
monofunctional alkylating agents (10). This severe DNA repair defect
appears to be the primary cause for the observed cytotoxicity of
N-methyl-N-nitrosourea, methylmethanesulfonate (MMS), or It was assumed for many years that PARP activity was associated with a
single protein displaying unique DNA damage detection and signaling
properties. This assumption was challenged by the recent discovery in
Arabidopsis thaliana of a gene coding for a PARP-related
polypeptide of a calculated molecular mass of 72 kDa (11). It then
became evident that two structurally different PARP proteins, both
possessing DNA-dependent poly(ADP-ribose) activities, were
present in both A. thaliana as well as in maize (12-14).2 Furthermore, it
has been reported recently that mouse embryonic fibroblasts derived
from PARP knockout are capable of synthesizing ADP-ribose polymers in
response to DNA damage (15), suggesting that in mammals, like in
plants, at least one additional member of the PARP family may exist in
addition to the classical zinc finger containing PARP.
This work describes the cloning of a human and a murine cDNA coding
for a new member of the PARP family, based on its similarity with the
A. thaliana 72-kDa poly(ADP-ribose) polymerase (12), which
those authors named APP. We denote this new protein PARP-2, to
differentiate from the classical PARP protein renamed PARP-1. We
demonstrate that PARP-2 is a nuclear DNA-dependent
poly(ADP-ribose) polymerase catalyzing the formation of ADP-ribose
polymers in the presence of damaged DNA, thus suggesting a biological
role in the cellular response to DNA damage.
Determination of Poly(ADP-ribose) Synthesis in Cell
Extracts--
To determine poly(ADP-ribose) synthesizing activity in
cells extracts, equal amounts of protein from various cell extracts were incubated in standard conditions with 200 µM
[ EST Searches and Cloning of cDNA Encoding Murine
PARP-2--
Sequence analyses were performed using the GCG sequence
analysis package (Wisconsin package version 8.1, Genetic Computer Group, Madison, WI). The primary sequence of the A. thaliana
PARP homologue (APP, GenBank 228 accession number Z48243) was used to
search the complete EST data base (release 107/55 of GenBank 228 data
base) using TBLASTN of the BLAST program (16). Two murine and one human
EST (GenBank 228 accession numbers AA608364, AA529426, and AA595596,
respectively) encoding APP homologues but distinct from the classical
PARP-1 of 113 kDa were chosen and purchased from the IMAGE consortium
(Research Genetics, Huntsville, AL). EST clone number AA529426
contained the cDNA (1.2 kilobase) encoding the catalytic domain of
mouse PARP-2 (aa 199-559) in the pCMV SPORT vector (Research Genetics,
Huntsville, AL). In order to express the catalytic domain of PARP-2, a
SmaI site and an ATG surrounded by the translation
initiation sequence (17) of PARP-1 (18) were created by PCR, using the
sense primer AGGCCCCGGGGGGAGGATGGCGACTTTGAAGCCTGAGTCTCAG and the
reverse primer CCAGCAAGGTCATAGTATAGTCC. The PCR fragment (550 bp) was
restricted with SmaI-AccI, and used to replace
the wild type sequence in clone AA529426. The entire catalytic fragment
(homologous to domain F in Fig. 3) was thereafter excised with
SmaI-NotI and cloned in the baculovirus
recombination vector pVL 1393, giving pVL-mPARP-2-F.
In order to generate a full-length mPARP-2 cDNA, a
SalI-NotI fragment of AA529426 was used to screen
a random-primed
A human PARP-2 EST clone was identified in the LifeSeqTM
data base of Incyte Pharmaceuticals using the human PARP-1 sequence for
a data base search. The clone (2286233) was ordered and analyzed. It
started 608 bp downstream of the start codon and ended 130 bp
downstream of the stop codon. A digoxigenin-labeled probe (digoxigenin labeling kit, Roche Molecular Biochemicals, Mannheim, Germany) derived
from this clone was used to screen a human total brain cDNA library
(CLONTECH, Palo Alto, CA). Several overlapping
positive clones were isolated and sequenced. One of them had an insert length of 857 bp starting 2 nucleotides upstream of the start codon and
covering 855 bp of the human PARP-2 open reading frame. These clones
were used to construct a full-length human PARP-2 cDNA.
Fluorescence in Situ Hybridization (FISH) Analysis--
Human
chromosomes were prepared from human peripheral blood lymphocytes
immediately after incorporation of bromodeoxyuridine. Mouse chromosomes
were prepared from normal mouse fibroblast cultures. The mouse
PARP-1 gene was identified with a 5-kilobase clone (of the
mouse PARP-1 gene), which was labeled by nick-translation with biotin-11-dUTP (Sigma, France). The PARP-2 gene was
identified in the mouse by a probe biotinylated by PCR consisting of
the full-length gene; and in the human by a 1.3-kilobase clone of human
PARP-2 cDNA, namely, EST AA595596, which was labeled by
nick-translation with biotin-11-dUTP (Sigma, France). A standard hybridization procedure was used as described previously (19). The
mouse PARP-1 probe was used at a concentration of 15 ng/ml in the
presence of a 100-fold excess of mouse Cot-1 DNA; the human PARP-2
probe was used at a concentration of 20 ng/ml and the PCR probe of
mouse PARP-2 at a concentration of 1 ng/ml in presence of 600-fold
excess of mouse Cot-1 DNA, in 15 ml of hybridization buffer for each
slide. Direct banding of bromodeoxyuridine-substituted human
chromosomes was obtained by incubation in an alkaline solution of
p-phenylenediamine (PPD11) (20) and staining with propidium iodide. Mouse chromosomes were stained with DAPI and identified by
computer-generated reverse DAPI banding. Immunochemical detection of
hybridization was performed using goat anti-biotin antibodies (Vector
laboratories, Burlingame, CA) and rabbit FITC-conjugated anti-goat
antibodies (Biosys, Compiègne, France). Metaphases were observed
under a fluorescent microscope (DMRB, Leica, Germany). Images were
captured using a cooled photometrics CCD camera and Quips-smart capture
software (Vysis).
RNA Preparation and Northern Blot
Analysis--
Poly(A)+ mRNA was purified from
PARP-1+/+ and PARP-1 Generation of Antibodies against PARP-2--
Two rabbits were
immunized by intramuscular injections of 200 µg of purified mPARP-2
catalytic domain (aa 204-559) in the presence of complete Freund's
adjuvant for the first inoculation (day 0) and incomplete Freund's
adjuvant for subsequent inoculations on days 15, 30, 45, and 60. The
rabbits were bled every fortnight until week 14, beginning a week after
the second injection. This antibody named YUC, raised against the
PARP-2 catalytic domain, recognizes both human and murine PARP-2, but
not PARP-1.
Immunofluorescence Microscopy--
Immortalized mouse embryonic
fibroblasts (3T3 cells) derived from PARP-1 Cloning and Expression of Nter-PARP-2 Fused to GST or GFP--
A
180-bp EcoRI-EcoRI fragment corresponding to aa
1-69 of mouse PARP-2, and named mouse Nter-PARP-2, was generated by
PCR from pVL-mPARP-2-F with the 5'-oligonucleotide
GGATCCCGGGAATTCGGATGGCGCCGCGGCGGC and the 3'-oligonucleotide
GGCTTTGCCCGAATTCTTTAACAGCAAGGTCT and cloned either into pGEX-2T
expression vector or into pEGFP-C3 in-frame with the GST or GFP reading
frame, respectively. The GST fusion protein was overexpressed and
purified using glutathione-Sepharose 4B (Pharmacia Biotech Inc.)
according to the specifications of the manufacturer. The
pEGFP-Nter-PARP-2 construction was used to transfect HeLa cells, and
transient expression of the GFP-Nter-PARP-2 fusion was monitored by
fluorescence microscopy, 24-48 h after transfection.
Overproduction and Purification of the Murine Recombinant
PARP-2--
pVL PARP-2 and the Baculogold 228 linearized baculovirus
DNA (Pharmingen) were co-transfected into Sf9 cells
according to the manufacturer's instructions. Cell propagation and
protein production was performed as described previously (22). The
identity of the purified protein was confirmed by Western blot with the polyclonal antibody against PARP-2 (1:2000 dilution). Purification of
PARP-2 was performed as described previously (23) for the purification
of chicken PARP-1 catalytic domain with some modifications. Briefly,
the cell pellet (1.5 × 109 cells) was homogenized in
75 ml of 100 mM Tris-HCl, pH 7.5, 0.2% Tween 20, 0.2%
Nonidet P-40, 14 mM Western Blot, Southwestern Blot, and Estimation of Amount of
hPARP-2--
Immunoblots and Southwestern blot were carried out as
described previously (25). The number of PARP-2 molecules per cell was
estimated in a Western blot experiment by comparing the intensity of
the immunoreaction of anti-PARP-2 antibody on PARP-2 (YUC) from 400,000 HeLa cells with increasing amounts of purified PARP-2.
Stimulation by DNA Strand Breaks--
Samples were incubated for
10 min at 25 °C in assay buffer (100 µl) consisting of 50 mM Tris-HCl, pH 8.0, 4 mM MgCl2,
0.2 mM dithiothreitol, with or without 200 ng of calf
thymus DNA previously treated with DNase I and 400 µM
[ Auto Poly(ADP-ribosylation) Reaction--
800 ng of purified
PARP-2 was incubated with 200 ng of calf thymus DNA previously treated
with DNase I or without DNA, in 100 µl containing 100 mM
Tris-HCl, pH 8.0, 10 mM MgCl2, 10 mM dithiothreitol, and 800 nM
[ Polymer Analysis--
To analyze the polymer synthesized by
either PARP-1 or PARP-2, samples were incubated in a standard enzymatic
assay with 400 µM
[ Determination of Kinetic Parameters--
Samples were incubated
for 10 min at 25 °C in assay buffer (100 µl) consisting of 50 mM Tris-HCl, pH 8.0, 4 mM MgCl2,
200 µM dithiothreitol, 200 ng of calf thymus DNA
previously treated with DNase I and various concentrations of
[ Poly(ADP-ribose) Synthesis in PARP
To confirm that poly(ADP-ribose) polymerase activity was present in
PARP-1
The reaction products synthesized in the presence of
[ Cloning of the Full-length Murine PARP-2 (mPARP-2)--
In plant
cell nuclei, PARP activity has been reported to be associated with a
protein of approximately 113 kDa (30). However, in A. thaliana, the cloning of a PARP homologue (11) revealed a protein,
called APP, with a theoretical Mr of 72,000. This protein showed a high similarity (60%) to the catalytic domain of
vertebrate PARP, but was completely divergent in its
NH2-terminal extremity where it harbored a helix-loop-helix
domain. The existence of two distinct PARP genes in plants
was definitely confirmed by the cloning of two different maize genes
coding for a classical PARP with zinc fingers of 110 kDa (ZAP) and a
structurally non-classical PARP (NAP, homologous to APP) of 72 kDa,
respectively (12, 14). Therefore, we undertook a search for the
mammalian equivalent of APP and NAP. Expressed sequence tags (EST) of
the GenBank/EMBL data bases (dbEST) were screened with the APP primary
sequence. Several EST from mouse and human were identified which shared homology with the catalytic domain of APP, while distinct from PARP-1.
These sequences were candidates for a new mammalian PARP homologue,
possibly responsible for the poly(ADP-ribosylation) activity detected
in PARP-1 Chromosomal Localization--
The chromosomal localization of the
human PARP-2 gene was identified by FISH on human
chromosomes using a human PARP-2 probe. Consistent signals on
chromosome 14, band 14q11.2 were identified (Fig.
3A); 100% of 25 metaphases
showed at least one signal on chromosome 14 at 14q11.2; 24% of the
metaphases showed signals on both chromosomes 14 on the same position.
Similarly, the murine PARP-2 gene was mapped using a murine
PARP-2 probe. FISH on mouse chromosomes exhibited consistent signals on
chromosome 14, band 14C1 (Fig. 3B); 70% of 25 metaphases
showed signals on 14C1; 10% had double signals on chromosome 14 band
14C1, and 20% showed signals on the two homologues at the same
position. As a control, chromosomal localization of the murine
PARP-1 gene was performed. FISH on mouse chromosomes
exhibited consistent signals on chromosomes 1, band 1 H5 (Fig.
3C); 30 metaphases were observed, of which 74% showed
signals in this position; 41% showed one signal on both chromosomes 1, 26% showed double signals on one of the two homologues. Altogether,
these results confirm the existence of two distinct and unique genes
coding for two different PARP molecules. A strict synteny was observed
between man and mouse in the two chromosome regions containing the
PARP-1 gene as well as those containing the
PARP-2 gene.
Expression of mPARP-2 mRNA--
Northern blot analysis
performed on poly(A)+ mRNAs of 3T3 cells derived from
PARP-1+/+ and PARP-1 Structural Organization of mPARP-2--
The complete nucleotide
sequence of murine PARP-2 predicts a full-length cDNA clone of
1,707 bp containing 16 bp of 5'-noncoding sequence which has been
confirmed by sequencing the 5' region of the genomic
DNA,3 a single open reading
frame of 1,680 bp, and a 3'-noncoding region of 125 bp. The deduced
amino acid sequence predicts a protein of 559 amino acids and a
molecular mass of 62 kDa. The amino acid sequences of human and murine
PARP-2s (hPARP-2 and mPARP-2, respectively) can be aligned with human
PARP-1 and the two plant PARP enzyme sequences APP and NAP (12, 14)
(Fig. 5A). The alignment shows that the carboxyl-terminal regions of PARP-2 are highly conserved in
comparison with the sequence of PARP-1 and the plant enzymes at their
catalytic domain (43% identity between hPARP-1 and hPARP-2 catalytic
domains). Moreover, it contains the PARP signature corresponding to the
ADP-ribose donor site (aa 401-450) in mPARP-2 as well as the crucial
residues forming the acceptor site (E534, K445, Y532, N448, H370, L530,
M432), including the catalytic glutamate at position 534 (31).
Interestingly, the blocks of sequence conservation (Fig. 5A)
correspond strictly to the secondary structure of the PARP-1 catalytic
domain, whereas, as frequently observed, sequence variability occurs
mainly in loops (data not shown). A short variable region (aa 195-207)
contains glutamate residues that may be potential polymer acceptors
sites. This new PARP is then expected to have similar catalytic
properties to PARP-1. It is noted that, the NH2-terminal
part of PARP-2 (aa 1-64) has no significant homology with any other
previous PARP. Interestingly, the NH2 terminus region of
human and mouse PARP-2 shows a higher sequence variability compared
with the highly conserved COOH terminus catalytic region (62% identity
between the NH2 terminus of mPARP-2 and hPARP-2) and no
homology with APP. The catalytic domains of mPARP-2 and hPARP-2 or APP
share 87 or 47% identity, respectively. However, this region contains
basic residues that could bear potential DNA-binding properties. On the
other hand, these basic residues could be involved in the nuclear
and/or nucleolar targeting of the protein (32). Therefore the
structural organization of PARP-2 is reminiscent of PARP-1, in the
sense that it is modular, made up of two distinct domains, one
responsible for the catalytic function, the other involved in putative
DNA binding function (Fig. 5B).
Overproduction and Purification of the Recombinant
mPARP-2--
Overexpression of full-length mPARP-2 and its catalytic
domain was carried out in baculovirus. This generated polypeptides of
62 and 40 kDa, respectively. These apparent molecular masses corresponded with the predicted values deduced from the respective cloned mPARP-2 cDNA. These proteins were purified following a procedure similar to that used for chicken PARP-1 overexpressed in
baculovirus (23) (Fig. 6, A
and B). Typically, 1 liter of cell culture yielded 10 mg of
highly purified 40-kDa catalytic domain or 62-kDa full-length mPARP-2.
The purified catalytic domain was used to raise a polyclonal antibody
(YUC) that did not cross-react with PARP-1 despite the high homology
between their catalytic domains (data not shown). A Western blot
analysis using this antibody showed that the apparent molecular weight
of the purified full-length mPARP-2 was identical to that of the
endogenous protein from 3T3 cell extract (Fig. 6C). This
result demonstrated that the size of the endogeneous protein of 3T3
cells is identical to the protein encoded by the open reading frame
from mPARP-2 cDNA. In addition, the abundance of PARP-2 in 3T3 and
HeLa cells was determined (see "Experimental Procedures") and
indicated about 200,000 molecules per cell (data not shown) which is
comparable to that of PARP-1 (33, 34).
Nuclear Location of mPARP-2--
In PARP-1-deficient 3T3 cells,
the poly(ADP-ribose) synthesized after DNA damage is found in the
nucleus (Fig. 1) favoring a nuclear location for the PARP(s) involved
in that process. The cellular distribution of mPARP-2 was addressed by
immunofluorescence using the polyclonal anti-mPARP-2 antibody (YUC) in
PARP-1 mPARP-2 Binds To and Is Activated by DNase I-damaged DNA--
The
activity of PARP-1 is dependent on the presence of DNA. To test the
hypothesis that PARP-2 shares a similar property, purified protein was
incubated with [
Since, in all the previously known PARP-1 proteins the DNA-binding
function is harbored in a domain distinct from the catalytic domain, we
hypothesized that the NH2-terminal part of PARP-2 might contain a DNA-binding property. Thus, the NH2-terminal
domain of mPARP-2 (aa 1-69) was expressed as a fusion product with GST (GST-NterPARP-2) in Escherichia coli, purified, and tested
for its DNA binding capacity by Southwestern analysis (Fig.
8B). Both the purified full-length mPARP-2 and the
GST-NterPARP-2 proteins bound 32P-labeled DNA activated by
DNase I confirming that PARP-2 is a DNA-binding protein and that aa
1-69 are sufficient for this function, in addition to the function of
a nuclear location signal. Thus, this small domain carries two features
important for the biological role of PARP-2: targeting to the nucleus
and binding to DNA.
Autopoly(ADP-ribosylation) of mPARP-2--
To demonstrate the
ability of mPARP-2 to synthesize ADP-ribose polymers, the
autopoly(ADP-ribosylation) of mPARP-2 was examined by following the
electrophoretic mobility of the enzyme incubated with
[
To characterize the product of reaction, the radioactivity associated
with the protein was resolved on a 20% denaturating polyacrylamide
gel. The "ladder" characteristic of ADP-ribose polymers was
observed for mPARP-2 and the polymer lengths were comparable to that of
hPARP-1. The structure of the product was further characterized by
two-dimensional TLC following polymer digestion by snake venom (28).
The three products expected, AMP, PRAMP, (PR)2AMP, were
obtained indicating the similarity between the polymer synthesized by
mPARP-2 and hPARP-1 in terms of size, chain length, and branching
frequency (29).
Kinetic Parameters of mPARP-2--
To compare the catalytic
properties of mPARP-2 with that of hPARP-1, their enzymatic activities
were assayed under the standard conditions. Analysis by the
Lineweaver-Burk plot estimated a Km of 130 µM for mPARP-2, which represents an affinity for
NAD+ 2.6-fold lower than hPARP-1 (50 µM). A
kcat of 42 × 10 In this work, which was initiated to resolve the intriguing
persistence of poly(ADP-ribose) formation in PARP-1 PARP-2, together with the plant enzymes APP and NAP, belongs to a class
of PARP molecules characterized by (i) a short, variable and basic
NH2-terminal domain, ranging from 64 in PARP-2 to 140 residues in length in APP and NAP, bearing both the DNA-binding element
and a nuclear location signal and (ii) a highly conserved COOH terminus
domain highly homologous to the PARP-1 catalytic domain.
The alignment of the sequences shows that in the catalytic domain all
the residues crucial for the activity (initiation, elongation, and
branching) are conserved suggesting that the catalytic function should
also be conserved in PARP-2. Indeed, the analysis of the enzyme
products demonstrated this functional conservation between both enzymes
(PARP-1 and PARP-2). Looking for protein substrates of PARP-2, we
noticed that purified histones were not polymer acceptors in
heteromodification reactions (data not shown). However, we cannot rule
out the existence of physiological acceptors for PARP-2. PARP-2
automodifies itself and possesses glutamate residues that could play
the role of acceptor sites. In PARP-1 the ADP-ribose acceptor sites are
mainly located in the automodification domain D. Although this domain
is absent in PARP-2, automodification takes place efficiently,
indicating that this mode of regulation has also been conserved. In
PARP-1, domain D contains a BRCT motif (36-38) that is, together with
domain A, the major protein-protein contact interface for interaction
with partners (8, 39). The absence of these motifs in PARP-2 suggests
that if it interacts with protein partner(s), this interaction should
be driven by some other mechanisms involving a binding module that
still has to be discovered.
Perhaps the most intriguing feature of PARP-2 is its binding to DNA and
its activation by DNA that has been treated with DNase I, despite the
absence of the characteristic zinc finger module which acts as a nick
sensor in PARP-1 (5, 40). The DNA-binding domain in mPARP-2 encompasses
aa 1-64 but does not present any obvious DNA binding motif. However,
it is rich in basic amino acids (27% Lys or Arg), which are likely to
be involved in this function. Given that DNA treated with DNase I
contains a complex mixture of DNA ends, footprinting experiments with
defined DNA probes will be necessary to further understand the
DNA-dependent activation of PARP-2. Even though, if PARP-1
and PARP-2 respective DNA-binding domains interact in a different
manner with different DNA structures, we may speculate on a possible
conservation of the activation mechanism based presumably on a
conformational change of the active site loop induced by DNA binding
(31).
Despite major differences between PARP-1 and PARP-2 including the
smaller size, the absence of zinc fingers and BRCT domains, PARP-2 like
PARP-1 is targeted to the nucleus, binds to and is activated by DNA
which has been treated by DNase I which in turn stimulates
poly(ADP-ribose) synthesis. While the catalytic function is
structurally and mechanistically conserved between the two enzymes,
their physiological role most probably differs. Their functional
specificity is presumably determined by their variable NH2-terminal modules.
While this work was in progress, a third member of the PARP family,
tankyrase, was identified and localized to human telomeres (41).
Tankyrase is a 142-kDa protein having similarity to ankyrins and to
PARP-1 catalytic fragment. Tankyrase is able to synthesize poly(ADP-ribose) but apparently independently of the presence of DNA.
TRF1 which is a negative regulator of telomere maintenance can be
modified in vitro by tankyrase. Again, one can imagine that
the function of tankyrase on telomeres is regulated by modules distinct
from the catalytic region.
The existence of a family of PARP proteins raises a number of important
questions with regards to their specific functions. We cannot exclude
the possibility that they could function as backups in the same cell
survival pathway. One has also to realize that the global PARP activity
measured in a damaged cell may in fact represent the addition of at
least two distinct enzymatic activities (PARP-1, PARP-2, and perhaps
more). The disruption of the PARP-2 gene will be necessary
to elucidate both its physiological role during DNA damage and repair,
and a possible functional redundancy.
All these new PARP proteins share a similar catalytic site and were
shown to be inhibited by 3-aminobenzamide (41, 42) (this work, Fig.
2A). This common inhibition may explain some "side effects" while
attempting to inhibit PARP-1 with inhibitors that were thought to be
specific for this enzyme, possibly by interfering with different
biological functions related to the other PARP. Under these conditions,
the pharmacological inhibition of PARP activity under pathological
conditions will certainly require detailed understanding of the
specific role of the different PARP family members in vivo
as well as detailed crystal structures of their catalytic sites.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-rays leading to cell death occurring after a
G2/M block (10).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]NAD+ (30.0 nCi/nmol), 2 µg/ml
histones, and 10 µg/ml DNA activated by DNase I at 25 °C for 10 min. Activity is expressed as the ratio between the radioactivity of
the acid insoluble material produced by PARP-1+/+ and
PARP-1
/
cell extracts.
ZAP II cDNA library (kindly provided by J. M. Garnier, IGBMC, Illkirch, France) established from 10-day-old mouse
embryos. One of the positive clones selected contained part of the
murine PARP-2 cDNA, starting 45 bp upstream of the ATG and encoded
aa 1 to 527 of the protein. In order to obtain good expression in the
baculovirus expression system, the region around the ATG was replaced.
A SmaI site and an ATG surrounded by the translation
initiation sequence of PARP-1 were introduced by PCR. The primers used
were, sense primer: AGGCCCCGGGGGGAGGATGGCGCCGCGGCGGCAGAGATCAGGCTCTGG,
reverse primer: ATCATCATTTCTTCCATGG. After purification, the PCR
product (712 bp) was restricted by SmaI-NcoI and
cloned in pVL-mPARP-2-F, to generate the full-length mPARP-2
baculovirus recombination vector pVL-mPARP-2 vector. The amplified
fragments were sequenced to ensure that no mutation has been introduced
by the PCR.
/
3T3 cells using the
messenger RNA isolation kit from Stratagene (La Jolla, CA). Total mouse
tissues RNA was purchased from Ambion (Austin, TX). Two micrograms of
poly(A)+ mRNA and 10 µg of total RNA were
fractionated on 1% agarose, 2.2 M formaldehyde gels and
transferred to Hybond N nylon membrane (Amersham). The murine PARP-2
probe corresponded to the 800-bp internal EcoRI fragment.
The blot was hybridized for 16 h with probe labeled by the random
priming method (1 × 106 cpm/ml) (21) in ExpressHyb
mixture (CLONTECH, Palo Alto, CA), washed, and
autoradiographed at
80 °C for 48 h.
/
and
PARP-1+/+ mice (5) and HeLa cells were grown on coverslips.
For PARP activity experiments, cells were exposed either to 1 mM hydrogen peroxide (Sigma) for 10 min or 1 mM
MMS (Aldrich) for 30 min and fixed with methanol/acetone (1/1, v/v) for
10 min at 4 °C and washed three times with phosphate-buffered saline
supplemented with Tween 0.1% (v/v). Cells were incubated overnight at
4 °C with a monoclonal anti-poly(ADP-ribose) antibody (H10) (1:200 dilution) or with the anti-PARP-2 polyclonal antibody (1:1000 dilution). After washing, the cells were incubated for 4 h at room
temperature with a 1:400 dilution of FITC-conjugated anti-mouse or
Texas Red-conjugated anti-rabbit antiserum. Immunofluorescence was
evaluated using a Zeiss Axioplan equipped with a C5985 chilled CCD
camera (Hamamatsu).
-mercaptoethanol, 10 mM
EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 M
NaCl and an anti-protease mixture (complete 228, Mini, Roche Molecular
Biochemicals). After sonication, (1 min at 30% of the maximum output
using a Branson SONIFIER large probe), the cell lysate was cleared by
centrifugation at 50,000 × g for 90 min. After
precipitation with protamine sulfate (1 mg/ml), and clarification by
centrifugation at 50,000 × g for 25 min, the proteins
precipitating at 35% ammonium sulfate saturation were eliminated by
centrifugation (20,000 × g for 20 min). Further ammonium sulfate was added to the supernatant to 70% saturation and
the pellet was collected after a 20-min centrifugation at 20,000 × g. The proteins were resuspended in 150 ml of 100 mM Tris-HCl, pH 7.5, 14 mM
-mercaptoethanol,
10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride
containing the antiprotease mixture. This sample was subjected to a
3-aminobenzamide Affi-Gel 10 column chromatography. The elution was
performed with 3-methoxybenzamide and fractions containing the
polypeptide were concentrated on an ultrafiltration membrane Diaflo
YM30 (Amicon, Inc.). A sample of each step of the purification was
stored for further analysis on SDS-PAGE and quantification of proteins
by the method of Bradford (24).
-32P]NAD+ (100 nCi/nmol). The reaction
was stopped by the addition of 5% (w/v) trichloroacetic acid
containing 1% (w/v) inorganic phosphate, and the acid-insoluble
radioactivity was washed 3 times in the same solution and once in 95%
EtOH and the radioactivity was measured. The stimulation of PARP
activity by DNA treated with DNase I was expressed as the ratio of the
activity of PARP with activated DNA to the activity of PARP without DNA.
-32P]NAD+ (100 nCi/nmol) (26). After the
indicated time of incubation at 25 °C, the reaction was stopped with
ice-cold acetone (80% v/v) and incubated for 30 min at
20 °C.
Insoluble material was pelleted by centrifugation for 20 min at
4 °C, washed once with 100% acetone, once with water-saturated
ether, and dried. The pellet was resolubilized in 50 µl of 1 × Laemmli buffer and analyzed on 8% SDS-PAGE. Gels were stained with
Coomassie Blue, destained, dried, and autoradiographed on Kodak Bio-Max
MS film.
-32P]NAD+. Radioactive proteins were
treated with 100 mM NaOH, 20 mM EDTA for 1 h at 60 °C (27). The solution was then neutralized with 100 mM HCl. One volume of phenol/chloroform (1:1) was added and remaining traces of phenol/chloroform were extracted from the aqueous
layer three times with diethyl ether. The polymer was then precipitated
twice with ethanol and the pellets were dissolved in water. The polymer
was either analyzed on a sequencing gel or treated with snake venom
phosphodiesterase and analyzed by two-dimensional thin-layer
chromatography according to Keith et al. (28). The
radioactive spots on the TLC were scraped from the thin layer and
32P label was determined by scintillation counting. The
average polymer size and the branching frequency were calculated
according to Miwa and Sugimura (29).
-32P]NAD+ (100 nCi/nmol). The reaction
was stopped by the addition of 5% (w/v) trichloroacetic acid,
containing 1% inorganic phosphate, and the acid-insoluble
radioactivity was washed 3 times in the same solution and once in 95%
EtOH and the radioactivity measured.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
Mouse Embryonic
Fibroblasts--
In a previous paper (5) we described the inactivation
of the PARP-1 gene, in mouse by homologous recombination.
Gene disruption was assessed by Southern blotting and by Western
blotting using a panel of specific monoclonal and polyclonal antibodies
that failed to detect the full-length PARP-1 or any of its functional NH2-terminal or COOH-terminal domains. Using low doses of
damaging agents (H2O2 or MMS) that are known to
trigger PARP-1 activity in wild-type (wt) cells, no ADP-ribose polymers
were detected in PARP-1
/
cells (data not shown).
However, high doses of damaging agents were able to trigger
poly(ADP-ribose) synthesis in PARP-1+/+ cells (Fig.
1, G) as well as in
PARP-1-deficient cells (Fig. 1, C and E), as
shown by immunofluorescence. This result suggests that there is a
poly(ADP-ribose) polymerase activity distinct from PARP-1, that is
activated by DNA damage.
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Fig. 1.
Immunofluorescence analysis of
poly(A)DP-ribose formation in PARP-1+/+ 3T3 (G
and H) and PARP-1 /
3T3 cells
(A-F). Cells were mock exposed (A and
B) or exposed to 1 mM
H202 for 10 min (C and
D), or to 1 mM MMS for 30 min (E-H).
Poly(ADP-ribose) was detected with the 10H monoclonal antibody (43)
followed by the fluorescein isothiocyanate-conjugated anti-mouse
antibody (A, C, E, and G). Nuclei were stained
with DAPI (B, D, F, and H).
/
cells and evaluate its contribution, a
quantitative and qualitative analysis of the reaction products was
performed. Whole cell extracts were prepared from spleen, testis,
primary or 3T3 embryonic fibroblasts of PARP-1+/+ and
PARP-1
/
mice, and tested for PARP activity. The results
displayed in Fig. 2A show that
all PARP-1
/
cells tested display 5 to 10% of total
PARP activity stimulated by DNA strand breaks, compared with wt cells.
This residual activity is inhibited by 2 mM
3-aminobenzamide supporting the idea of a new PARP enzyme activity.
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Fig. 2.
Characterization of residual poly(ADP-ribose)
synthesis in PARP-1 cell extract. A, relative PARP
activity in cells from spleen, testis, primary mouse embryonic
fibroblasts (MEF), or 3T3 fibroblasts isolated from
PARP-1+/+ and PARP-1 /
mice. Cell extracts
were incubated in standard conditions with 200 µM
[
-32P]NAD+ (30.0 nCi/nmol), 2 µg/ml
histones, and 10 µg/ml DNA activated by DNase I at 25 °C for 10 min. Activity is expressed as the ratio between the radioactivity of
the acid-insoluble material produced by PARP-1+/+ and
PARP-1
/
cell extracts. B, size distribution
of ADP-ribose polymers synthesized by PARP+/+ and
PARP
/
3T3 cells on a 20% denaturating polyacrylamide
gel (XC, xylene cyanol).
-32P]NAD+ were characterized by removing
the radiolabeled material from the acceptor proteins and fractionating
by electrophoresis on long 20% denaturating polyacrylamide gels. The
distribution of ADP-ribose polymers synthesized in wt and
PARP-1
/
cells are similar (Fig. 2B). The
reaction products were further characterized by two-dimensional TLC
after polymer hydrolysis by snake venom phosphodiesterase (28). No
significant differences were observed in the products, PRAMP,
(PR)2AMP, and AMP synthesized by each cell genotype (data
not shown). These results demonstrate that PARP-1
/
cells exhibit a bona fide poly(ADP-ribose) polymerase
activity presumably associated with novel PARP protein(s), confirming a previous report by Shieh et al. (15).
/
cells. The murine EST were used to screen a
mouse ES cell cDNA library. We thus were able to construct the
complete cDNA (GenBankTM accession number AJ007780).
Simultaneously, the cloning of the homologous human cDNA (GenBankTM
accession number AJ236912) was undertaken following a similar strategy.
We propose to name this new gene poly(ADP-ribose) polymerase-2
(PARP-2).
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Fig. 3.
Chromosomal mapping of hPARP-2,
mPARP-2, and mPARP-1 genes. FISH of a
hPARP-2 on a human lymphocyte chromosome spread (A). FISH
analysis of mPARP-2 (B and B') and mPARP-1
(C and C') on mouse fibroblast chromosome
spreads. Human chromosomes are counterstained with propidium iodide (in
red) and mouse chromosomes are counterstained with DAPI (in
blue).
/
mice and on total
RNAs from mouse tissues (Fig. 4) revealed
a PARP-2 transcript at an apparent molecular size of 2.0 kilobase. The
same amount of transcript was detectable in both PARP-1+/+
and PARP-1
/
cell lines, indicating that there is no
compensation for the PARP-1 deficiency in PARP-1
/
cells
by up-regulation of PARP-2 gene expression. The tissue distribution of PARP-2 showed at least a basal expression in all tissues and higher expression in germline. Moreover, Northern blot
analysis on total mRNA from HeLa cells treated or untreated with
genotoxic agents such as UV-B (500 J/m2), UV-C (20 J/m2),
N-methyl-N'nitro-N-nitrosoguanidine
(50 µM), and H2O2 (0.5 mM) revealed that the level of PARP-2 mRNA is not
increased following genotoxic stress (data not shown), like for
PARP-1.
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Fig. 4.
Northern blot analysis of the murine
PARP-2 gene expression. Poly(A)+
mRNA isolated from PARP-1+/+ and
PARP-1 /
3T3 cells and total mouse tissues RNAs were
fractionated on 1% agarose gel in the presence of formaldehyde and
ethidium bromide and transferred on Hybond N filter (see
"Experimental Procedures"). Upper panel, hybridization
with the mPARP-2 probe. Lower panel, the loading and the
transfer of RNAs on filter are controlled under UV light to visualize
the 18 S and 28 S ribosomal RNAs stained with ethidium bromide
(EtBr).
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Fig. 5.
Sequence alignment of different PARP-2s with
hPARP-1 and schematic representation of the functional domains of
hPARP-1 and mPARP-2. A, sequence alignment of human
PARP-1 (hPARP-1, accession number P09874 (44-46)), human PARP-2
(hPARP-2, AJ236912, this work), murine PARP-2 (mPARP-2, AJ007780, this
work), Zea mais NAP (AJ222588) (14), and A. thaliana APP (Z48243) (11). Black and gray
boxes represent conserved or similar amino acids in at least four
out of five sequences, respectively. B, schematic
representation of the modular organization of human PARP-1 and mouse
PARP-2.
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Fig. 6.
Purification of the recombinant full-length
mPARP-2 and its catalytic domain overexpressed in baculovirus.
A, purification of mPARP-2 catalytic domain. Samples at
various stages of purification (see "Experimental Procedures") were
separated by 12% SDS-PAGE and the proteins were stained by Coomassie
Blue. B, purification of full-length mPARP-2. Samples were
analyzed as in A on 10% SDS-PAGE. C, Western
blot analysis of purified full-length mPARP-2 (a) and 3T3
cell extract (b) using the polyclonal anti-mPARP-2 antibody
(YUC).
/
3T3 cells. As expected PARP-2 accumulated in
the cell nucleus with an apparent peripheral location (Fig.
7A). In the
NH2-terminal part of PARP-2, the presence of basic motifs
could be responsible for nuclear targeting of the protein. To address
this possibility, aa 1-69 were fused to GFP and the fusion protein was
expressed in HeLa cells. While GFP is cytoplasmic (Fig. 7C)
the addition of aa 1-69 of mPARP-2 is sufficient to target the fusion
protein to the nucleus (Fig. 7E). This confirmed that the
nuclear location module of mPARP-2 is in a domain distinct from the
catalytic domain and located in the NH2-terminal part of
the protein, similar to the structure previously described in PARP-1
(35).
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Fig. 7.
Subcellular distribution of mPARP-2.
Localization of mPARP-2 in PARP-1 /
3T3 cells
(A and B) by immunofluorescent microscopy
analysis using the YUC polyclonal anti-mPARP-2 antibody and a Texas
Red-conjugated anti-rabbit antibody (magnification: × 63).
Localization of GFP (C and D) or GFP-NterPARP-2
(E and F) transiently overexpressed in HeLa
cells. The intrinsic fluorescence of both proteins were visualized by
fluorescent microscopy (magnification: ×40). B, D, and
F, DAPI or Hoechst staining of the nuclei.
-32P]NAD+ in the absence
or presence of calf thymus DNA treated with DNase I. Fig.
8A shows that the activity of
PARP-2 is stimulated by a factor of 15, while PARP-1 incubated in the
same conditions, is stimulated by a factor of 50. Even though the
activation of PARP-2 is lower than for PARP-1, this result shows
clearly that PARP-2 is also activated by DNA which has been treated by
DNase I.
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Fig. 8.
mPARP-2 binds to and is activated by
DNA. A, comparison of the activation of purified
hPARP-1 and mPARP-2 by DNA activated by DNase I. Activation factor
represents the ratio between the activity measured in the presence or
absence of DNA. Under these conditions 140 and 69 nmol/min of
ADP-ribose were incorporated per mg of PARP-1 and PARP-2, respectively.
B, Southwestern blot analysis of purified full-length
mPARP-2 (a and a'), GST-NterPARP-2 (b
and b'), and GST (c and c'). Proteins
were separated by 12% SDS-PAGE and either stained by Coomassie Blue
(left panel) or transferred onto nitrocellulose and detected
using 32P-labeled DNase I-activated DNA (right
panel).
-32P]NAD+ and DNA activated by DNase I. Fig. 9A shows the
autoradiographic profile of the PARP-(ADP-ribose) conjugates generated
at the indicated times. PARP-2 is efficiently automodified in the
presence of DNA strand breaks and the electrophoretic mobility of the
conjugates decreases with the time of incubation. This presumably
reflects the increase of the polymer size and complexity as the
incubation progresses. When the automodification reaction is performed
using higher NAD+ concentrations (close to the
Km value or above,) most of the resulting reaction
products remain concentrated in the wells of the gel (data not
shown).
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Fig. 9.
Analysis of the reaction products of purified
full-length mPARP-2. A, autopoly(ADP-ribosylation) of
purified mPARP-2 incubated with [ -32P]NAD+
in the absence (a) or presence of DNA activated by DNase I
(b-g) for the indicated times. Samples were separated by
10% SDS-PAGE and autoradiographed. The arrow represents the
electrophoretic mobility of purified mPARP-2 in the absence of NAD.
B, demonstration of a "ladder" synthesized by purified
hPARP-1 and mPARP-2 on a 20% denaturating polyacrylamide gel.
XC, xylene cyanol. BB, bromphenol blue.
C, two-dimensional thin-layer chromatography analysis of
product hydrolysis of polymer synthesized by purified full-length
mPARP-2. A branching frequency of 0.2 was found for an average polymer
size of 36 residues. These values are very closed to the values found
for an equivalent polymer size synthesized by PARP-1 (29).
PRAMP, phosphoribosyl-AMP; (PR)2AMP,
diphosphoribosyl-AMP.
3
s
1 was calculated assuming first-order kinetics, where
the only variable was the substrate NAD+, because PARP-2 is
both enzyme and second substrate. PARP-2 is then a much less efficient
enzyme in terms of catalysis than hPARP-1; the
kcat/Km ratio for mPARP-2 is
323 s
1 M
1 which is 18 times
lower than that of hPARP-1 (6,000 s
1
M
1). Thus, this activity measured
in vitro compares well with the 5-10% residual
activity found in PARP-1
/
cell extracts stimulated by
DNA strand breaks (Fig. 2A).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
cells, we have identified a new member of the PARP family, PARP-2. Human and mouse PARP-2 genes were mapped to chromosome
14q11.2 and 14C1, respectively, which are distinct from PARP-1 loci,
supporting the conclusion that PARP-2 is coded by a different gene.
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ACKNOWLEDGEMENTS |
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We are grateful to E. Flatter and G. de la Rubia for excellent technical assistance and Dr. S. Shall for critical reading of the manuscript. The 10H monoclonal anti-poly(ADP-ribose) antibody was a gift from Drs. Miwa and Sugimura. We acknowledge Incyte Pharmaceuticals for providing a partial human PARP-2 cDNA clone (ID2286233) identified in the LifeSeqTM Database.
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FOOTNOTES |
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* This work was supported in part by the Association pour la Recherche Contre le Cancer, Electricité de France, CNRS Grant ACC-SV Radiations ionisantes, Fondation pour la Recherche Médicale, and the Commissariat à l'Energie Atomique.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.
§ Contributed equally to the results of this report.
¶ Recipient of a postdoctoral fellowship from the Fondation pour la Recherche Médicale.
§§ To whom all correspondence should be addressed: UPR 9003 du CNRS, Laboratoire conventionné avec le Commissariat à l'Energie Atomique, Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard Sébastien Brant, F-67400 Illkirch, France. Tel.: 33-388-65-53-68; Fax: 33-388-65-53-52; E-mail: demurcia{at}esbs.u-strasbg.fr.
2 M. Kazmaier, personal communication.
3 C. Niedergang, J.-C. Amé, V. Schreiber, J. Ménissier-de Murcia, and G. de Murcia, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: PARP, poly(ADP-ribose) polymerase; MMS, methylmethanesulfonate; EST, expressed sequence tags; FISH, fluorescence in situ hybridization; PAGE, polyacrylamide gel electrophoresis; wt, wild type; aa, amino acid(s); PCR, polymerase chain reaction; bp, base pair(s); GST, glutathione S-transferase; GFP, green fluorescent protein; DAPI, 4,6-diamidino-2-phenylindole.
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REFERENCES |
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