Review articles
The PARP superfamily
Jean-Christophe Amé, Catherine Spenlehauer, and Gilbert de Murcia*
Summary
Poly(ADP-ribosyl)ation is an immediate DNA-damage-
dependent post-translational modification of histones
and other nuclear proteins that contributes to the survival
of injured proliferating cells. Poly(ADP-ribose) poly-
merases (PARPs) now constitute a large family of 18
proteins, encoded by different genes and displaying a
conserved catalytic domain in which PARP-1 (113 kDa),
the founding member, and PARP-2 (62 kDa) are so far the
sole enzymes whose catalytic activity has been shown to
be immediately stimulated by DNA strand breaks. A large
repertoire of sequences encoding novel PARPs now
extends considerably the field of poly(ADP-ribosyl)ation
reactions to various aspects of the cell biology including
cell proliferation and cell death. Some of these new
members interact with each other, share common part-
ners and common subcellular localizations suggesting
possible fine tuning in the regulation of this post-
translational modification of proteins. This review sum-
marizes our present knowledge of this emerging super-
family, which might ultimately improve pharmacological
strategies to enhance both antitumor efficacy and
the treatment of a number of inflammatory and neurode-
generative disorders. A provisional nomenclature is
proposed. BioEssays 26:882–893, 2004.
ß 2004 Wiley Periodicals, Inc.
Introduction
Forty years ago, in their initial paper entitled ‘‘Nicotinamide
mononucleotide activation of a new DNA-dependent poly-
adenylic acid synthesizing nuclear enzyme’’ Chambon, Weil
Unité 9003 du CNRS, École Supérieure de Biotechnologie de
Strasbourg, Illkirch, France.
Both authors contributed equally to this work.
Funding agency: Centre National de la Recherche Scientifique,
Association pour la Recherche Contre le Cancer, Électricité de
France, Ligue Nationale Contre le Cancer and Commissariat à
l’Énergie Atomique.
*Correspondence to: Dr. Gilbert de Murcia, Unité 9003 du CNRS,
École Supérieure de Biotechnologie de Strasbourg, Boulevard
Sébastien Brant, BP 10413, F-67412 Illkirch Cedex, France.
E-mail: demurcia@esbs.u-strasbg.fr
DOI 10.1002/bies.20085
Published online in Wiley InterScience (www.interscience.wiley.com).
Abbreviations: PARP, poly(ADP-ribose) polymerase; XRCC1, x-ray
cross complementing factor 1; BRCT, BRCA1 c-terminus; GFP, green
fluorescent protein; 3-AB, 3-aminobenzamide;; DAPI, 40 ,6-diamidino-
2-phenylindol; SSB, single strand breaks; SSBR, single strand breaks
repair; DSB, double strand breaks.
882 BioEssays 26.8
and Mandel correctly anticipated the major property of an
enzymatic activity involved in DNA-dependent NADþ con-
sumption. This seminal contribution triggered the science of
poly(ADP-ribose) (PAR) that today covers a large area of
biology dealing with genomic stability and energy metabolism.
Poly(ADP-ribosyl)ation of nuclear proteins is a post-transla-
tional modification induced by DNA strand breaks that
establishes a molecular link between DNA damage and
chromatin modification. A large body of evidence demon-
strates that this activity efficiently contributes to a detection–
signaling pathway leading ultimately to the resolution of
strand-break interruptions. In certain pathophysiological con-
ditions, this protecting function is strongly activated leading to
cell death, tissue damage and organ failure.
From genome sequencing and various molecular and
genetic approaches, it became evident that more than one
enzyme was involved in the conversion of NADþ to ADP-
ribose polymers. A superfamily of eighteen proteins containing
PARP domains has recently emerged. In this review, we have
summarized our present knowledge of this new family of
proteins, highlighting some of the yet unexplored functions of
PARP-1, its founding member.
The PARP superfamily: seven isolated cDNAs
The characterization of the human PARP family of proteins
was based on an exhaustive search of the non-redundant
protein database (NCBI) using the human PARP-1 catalytic
domain (GenBank XP_037275 residues 796–1014). Eighteen
different putative PARP homologues were found (Fig. 1) that
could potentially carry poly(ADP-ribose) polymerase activity.
Although, we are far from understanding the structure and
function of all members of the PARP family, biochemical and
enzymatic properties have been studied for at least seven of
them to various degrees, giving a provisional picture of this
rapidly growing area of post-translational modification of
proteins.
PARP-1, the founding family member, has been the most-
extensively studied. In response to DNA damage, it uses
NADþ to synthesize a linear or multibranched polymer of ADP-
ribose on various nuclear protein acceptors usually associated
with chromatin, or, itself in an automodification reaction. Both
automodification and heteromodification of acceptors con-
tribute positively to the survival of injured proliferating cells
(see for review(1,2)). The structure–function relationship of
PARP-1 is now well understood, four domains out of six
have been assigned a particular function: (1) domain A, the
DNA-binding domain (Fig. 1), containing two zinc finger
BioEssays 26:882–893, ß 2004 Wiley Periodicals, Inc.
 Review articles

Figure 1. The PARP superfamily. Schematic domain architecture of the eighteen members of the poly(ADP-ribose) polymerase (PARP)
superfamily. The proteins are drawn to scale and low-complexity segments in certain proteins have been omitted. Each protein has been
locally searched against the Pfam database version 7(35) using the program hmmpfam of the HMMER2 package. The most significant
domains detected have been indicated with colored boxes and their limits indicated by the position numbers. Here is a brief description of the
most significant one. The BRCT (BRCA1 C Terminus) domain is found predominantly in proteins involved in cell-cycle-checkpoint functions
responsive to DNA damage. The WWE domain is named after three of its conserved residues and is predicted to mediate specific protein–
protein interactions in ubiquitin and ADP ribose conjugation systems. The UIM motif is called the ubiquitin interaction motif. The SAP (after
SAF-A/B, Acinus and PIAS) motif is a putative DNA -binding domain found in diverse nuclear proteins involved in chromosomal organiza-
tion. The macro-H2A (A1pp) domain is found at the C terminus of the macro-H2A histone protein and is a predicted phosphoesterase
domain that is supposed to function as an ADP-ribose phosphoesterase that might regulate the protein(ADP-ribosyl)ation.(36) The RRM
motif is probably diagnostic of an RNA -binding protein. The dendrogram was based on the alignment of Fig. 2 and was generated with the
JALVIEW program.

structures responsible for PARP-1’s interaction with DNA
breaks, which are also found in DNA ligase III and in the 30 DNA
phosphoesterase from Arabidopsis thaliana (see for re-
view(3)), (2) domain B containing a bipartite nuclear localiza-
tion signal (NLS) responsible for the nuclear homing of PARP-
1 and a caspase-3 cleavage site,(4) (3) domain D, the
automodification domain, containing a BRCT motif constitut-
ing the major protein interface with various nuclear partners,
and (4) domain F, the smallest PARP-1 fragment retaining
catalytic activity (see for review(5)). The crystal structure of the
chicken PARP-1 catalytic domain(6) shows common structural
features with the active site of bacterial (ADP-ribosyl)ating
toxins (Diphteria toxin or Pertussis toxin) notably the NAD-fold:
b-a-loop-b-a corresponding, in PARPs, to the most conserved
region (PARP signature) (Fig. 2a,b). It contains an additional
a-helical domain (a-hd) that is thought to relay the activation
signal issued on binding to damaged DNA. Nothing is known
so far concerning the function of domains C and E.
The role of PARP-1 in facilitating DNA repair has been
clearly demonstrated by the generation of three independent
PARP-1-deficient mouse models (see for review(7)). These
animals showed hypersensitivity to ionizing radiation and

BioEssays 26.8 883

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Figure 2. The PARP signature. a: Multiple alignment of the eighteen human PARP sequences. The secondary structure of the domain as
determined from the chicken PARP catalytic domain(6) has been superimposed under the alignment with the following code: blue arrows for
b sheets, red cylinder for a helices and straight lines for loops. The amino acid color code is that of CLUSTALX(74) (blue: >60% hydrophobics
(ACFHILMVWY); Magenta: >50% negative charges (DE); Red: 60% positive charges (KR); Green: >50% polar (STQN); Pink: >85%
cysteines; Orange: >8% glycines; Yellow: >85% prolines; Cyan: >50% Aromatics (FYW)). The sequences are denoted by their common
laboratory name. b: Ribbon representation of the PARP signature.

alkylating agents at the cellular or whole-animal level.
Unexpectedly, these animals also showed protection against
various inflammatory processes such as cerebral and cardiac
ischemia and a higher resistance to septic shock (see for
reviews(2,8)).
PARP-2 was discovered as a result of the presence of
residual DNA-dependent PARP activity in embryonic fibro-
blasts derived from PARP-1-deficient mice.(9,10) Its catalytic
domain has the strongest resemblance to that of PARP-1 with
69% similarity. The crystal structure of the murine PARP-2 is
very similar to that of PARP-1 but with the exception of
differences in the vicinity of the acceptor site that reflect dif-
ferences in terms of substrates.(11) Clearly, PARP-2 prefer-
entially heteromodifies histone H2B (Huber et al., unpublished
results) whereas PARP-1 preferentially modifies histone
H1(12) in isolated nuclei. PARP-2 is a nuclear protein that
binds to and is activated by DNase-I treated DNA, however,
its DNA-binding domain differs from that of PARP-1 and it
targets DNA gaps but not nicks (J.C.A., unpublished data).
This domain, containing 75 aa, localizes the protein to the
nucleus and displays some homology with the SAP domain(13)
found in various nuclear proteins, such as AP-endonuclease
and Ku70, involved in chromosomal organization and in DNA
repair. Interestingly, this domain is also found duplicated in
plant PARP-2 orthologs (Arabidopsis thaliana #NP_192148
and Zea mays #T03656).
PARP-2 whose function is still not clear acts as a chromatin
modifier as mentioned above. PARP-2 interacts with PARP-1
and shares common partners involved in the Single Strand
Break Repair (SSBR) and Base Excision Repair (BER)
pathways: XRCC1, DNA polymerase-b, and DNA ligase III.
PARP-1 and PARP-2 also interact with proteins involved in the
kinetochore structure and in the mitotic spindle checkpoint
(see below). However, specific partners of PARP-2 are
beginning to be discovered, such as the telomeric protein
TRF2, suggesting a link with telomere integrity control.(14) The
involvement of PARP-2 in the cellular response to DNA
damage has been investigated by the generation of a PARP-2-
deficient mouse model.(15) Following treatment by the alkylat-
ing agent N-nitroso-N-methylurea (MNU), PARP-2-deficient
cells displayed a large delay in DNA-strand breaks resealing,
similar to that observed in PARP-1-deficient cells,(16) thus
confirming that PARP-2 is an active player in SSBR in spite of
its reduced capacity to synthesize ADP-ribose polymers.
However, PARP-2 deficient mice also display specific pheno-
types (see below), indicating that PARP-1 and PARP-2
functions are complementary but do not fully overlap.
PARP-3 has been identified as a core component of
the centrosome(17) preferentially located at the daughter
centriole throughout the cell cycle. Of all the PARPs, it dis-
plays the smallest N-terminal domain with only 54 residues
responsible for the centrosomal localization. PARP-3 shares

884 BioEssays 26.8


with PARP-1 two domains, E and F, which display a 61%
similarity. PARP-3 activity is hardly detectable but can never-
theless be inhibited by the PARP inhibitor 3-aminobenzamide.
PARP-3 interacts with PARP-1 which, in part, also resides in
the centrosome(18) suggesting a link between the DNA-
damage surveillance network and the mitotic fidelity check-
point (see below).
VPARP (PARP-4), the largest of the family (192.6 kDa),
was discovered associated with vault particles, a cytoplasmic
ribonucleoprotein complex that associates two other highly
conserved proteins, major vault protein (MVP) and telomer-
ase-associated protein (TEP1) and an untranslated vault
RNA (VRNA).(19) The function of these unusual particles is
unknown, but they may play a role in cellular transport and
could be associated with multidrug resistance in some cell
lines. The protein has also been localized to nuclear pores and
is associated with the mitotic spindle during mitosis. The
domain structure is quite unusual since domains E and F are
located at the N-terminal part of the protein. VPARP as well as
purified vault particles are capable of catalyzing the poly(ADP-
ribosyl)ation of MVP. PARP-4 contains a BRCT-motif-contain-
ing domain at the most N-terminal part of the protein and the
type A domain of the von Willebrand factor whose common
feature appears to be involvement in multiprotein complexes.
It has recently been shown that mVparp, alone or in
combination with mTep1, is dispensable for normal embryonic
development, telomerase catalysis, telomere length main-
tenance, and vault structure in vivo.(77)
Tankyrase 1(TRF1-interacting, ankyrin-related ADP-ri-
bose polymerase), (PARP-5a), was identified as a partner of
the human telomeric protein TRF1 in a two-hybrid screen-
ing.(20) It contains the smallest domain homologous to PARP-1
that still displays PARP enzymatic activity. The catalytic site
spans only 240 aa compared to 353 aa for PARP-1. The a-hd
domain (Fig. 1), comprising a succession of five N-terminal
helices in the PARP-1 crystal structure, is replaced in
Tankyrase by the SAM (Sterile Alpha Motif) domain also
made of a series of five helices arranged in a small globular
domain. This domain acts as a protein-interaction module
through its ability to homo- and hetero-oligomerize with other
SAM domains including Tankyrase 2.(21) The major part of the
protein, spanning 843 aa, is made of Ankyrin repeats orga-
nized in five clusters(22) involved in its interaction with partners.
In addition, the N-terminal domain contains homopolymeric
runs of His, Pro and Ser (HPS) harboring a MAP kinase
consensus motif (PXSP). The phosphorylation of this motif
upon insulin stimulation enhances Tankyrase 1 activity and
suggests that it is an important insulin signaling target.(23)
Overexpression of Tankyrase 1 promotes ADP-ribosylation of
TRF1, leading to its release from telomeres and to telomere
elongation(24) after which Tankyrase is finally ubiquitinated
and degraded by the proteasome.(25) The in vivo function of
Tankyrase 1 has been recently clarified by the Smith’s labo-
Review articles
ratory: shutting down Tankyrase 1 expression by siRNA blocks
the cells during early anaphase because sister telomeres are
unable to segregate, indicating that the replicated telomeres
are held together by specific cohesion complexes that are
resolved by Tankyrase 1.(26)
A second tankyrase (PARP-5b or tankyrase-2) encoded by
a distinct gene has also been discovered.(27) The sequence of
this protein exhibits >85% identity with tankyrase-1 but lacks
the HPS motif. Tankyrase 1 and Tankyrase 2 interact with the
same set of proteins (TRF1, IRAP, Glut4, Grb14, TAB 182 and
NuMa(28–33) and probably mediate overlapping functions in
telomere homeostasis and vesicle trafficking (glucose trans-
port and insulin signaling). Accordingly, Tankyrase 1 and
Tankyrase 2 show multiple subcellular localizations: telome-
res, nuclear pores, Golgi vesicles and pericentriolar material
(PCM) during mitosis. Tankyrase 2, however, displays distinct
properties from those of tankyrase 1. When overexpressed, it
induces caspase-independent cell death through the loss of
mitochondrial potential, a process inhibited by the PARP
inhibitor 3-aminobenzamide.(27)
TiPARP (PARP-7) was identified by differential display
as a 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-induced
mRNA.(34) The exact function of TiPARP remains unclear. It
seems to be involved in T-cell function, and its induction by
TCDD contributes to tumor promotion. The in vitro transcribed/
translated protein appears to be able to catalyze histone
poly(ADP-ribosyl)ation despite the absence of the catalytic
Glu residue equivalent to Glu 988 of PARP-1.
More PARPs from human
genome sequencing
To investigate further the possible biological role of the other
members of the PARP family, we looked at their potential
domain organization by searching the latest Pfam(35) database
using the HMMER hidden Markov model software (http://
hmmer.wustl.edu). The surprising feature of these proteins is
the diversity of the new domains that the PARP catalytic
domain is associated with, suggesting that they may be
implicated in many biological functions (Fig. 1). These proteins
are distinct from the previously described PARPs in that they
combine their catalytic domains with a variety of adapter
domains such as zinc fingers, SAP, Ankyrin, BRCT, Macro
domain and SAM.
The most-frequent domain found in five of the new PARPs
(PARP-7, PARP-11 to PARP-14) is the WWE domain occuring
in classes of proteins associated with ubiquitination.(36) The
link with ubiquitination is also found in the Ubiquitin Interaction
Motif (UIM) present in PARP-10. Three of the new PARPs
(PARP-7, PARP-8 and PARP-13) have one zinc finger and
PARP-12 has three zinc fingers that differ from the zinc finger
found in PARP-1 and in DNA-ligase III. The zinc fingers of
PARP-7, PARP-12 and PARP-13 are of the C-x8-C-x5-C-x3-H
type, which includes zinc finger proteins from eukaryotes
BioEssays 26.8 885
Review articles
involved in cell cycle or growth-phase-related regula-
tion. PARP-10 and PARP-15 have a motif specific for binding
to RNAs with the RNA Recognition Motif (RRM) found in
a variety of RNA-binding proteins, including heterogeneous
nuclear ribonucleoproteins (hnRNPs), proteins implicated
in regulation of alternative splicing, and protein compone-
nts of small nuclear ribonucleoproteins (snRNPs). This
motif also appears in certain single-stranded DNA-binding
proteins.
PARP-9 (bal) has recently been discovered in patients with
certain types of diffuse large B-cell lymphomas (DLBCL). It has
been described as a new nuclear protein encoded by the BAL
gene, which is expressed at a significantly higher level in fatal
high-risk DLBCLs and may be implicated in B-cell migra-
tion.(37) The overexpression of BAL increases the rate of
migration of B-cell lymphoma transfectants, suggesting that
the risk-related protein may promote the dissemination of high-
risk DBLCls. This protein contains a duplicated A1pp domain,
also called the macro domain, found in a number of otherwise
unrelated proteins. PARP-14 and PARP-15 also contain this
duplicated domain found at the C terminus of the macro-H2A
histone protein and in the non-structural proteins of several
types of ssRNA viruses. It is also found on its own in a family of
proteins from bacteria (P75918), archæbacteria (O59182) and
eukaryotes (Q17432), suggesting that it is involved in an
important and ubiquitous cellular process. The recent crystal-
lization of AF1521, a protein consisting of a stand-alone macro
domain from Archaeoglobus fulgidus shed some light on its
function.(38) Its structure closely resembles the N-terminal
DNA-binding domain of Escherichia coli leucine aminopepti-
dase PepA and shows some similarity to members of the
P-loop family of nucleotide hydrolases, like the phosphoes-
terases that act on ADP-ribose derivatives. Though it is
interesting to find the macro domain associated with potential
PARP domains, the sequence variation amongst the macro
domain sequences is so high compared to that of the AF1521
family that it is not clear if they still retain catalytic activity. It is
however possible that, in these proteins, the macro domain
functions as a nucleic-acid-binding module as is the case with
the PepA N-terminal domain.
These observations suggest that the new domains function
in specific interaction with other proteins and could help in
targeting the different kinds of poly(ADP-ribosyl)ation activ-
ities of proteins participating in various signaling cascades.
An interesting feature of the PARP family is the capacity of
some of its members to interact with each other, like PARP-1
with PARP-2(16) and PARP-3(17) and Tankyrase 1 with
Tankyrase 2.(28,32) This type of combination/association
could involve these complexes in different subcellular com-
partments and with different substrates, which they could
not otherwise target as individuals. This may highlight a new
organizational order for the PARPs that could diversify greatly
their biological responses, via combinatory interactions.
886 BioEssays 26.8
The PARP active site:
a conserved core in the family members
The sequence alignment relative to the 18 PARPs catalytic site
is presented in Fig. 2a. It reveals major conservation blocks
defining the ‘PARP signature’ that coincide with key secondary
structures constituting the active site. The highest sequence
and length variability is observed between positions 70 and
105 and between positions 168 and 202 of the alignment that
introduces some major gaps. The flexibility of these regions is
consistent with the crystal structure of the PARP domain(6) as
these regions represent loops of variable length that may act
as interacting sites to allow the recruitment of acceptor
proteins (Fig. 2b). The catalytic residue E988 of PARP-1 in
position 214 of the alignment has been found to be conserved
among PARP-1s from various species, in PARP-2, PARP-3,
PARP-4 and in Tankyrases (PARP-5a and b). This Glu residue
is also conserved in four of the new members: PARP-5c,
PARP-11, PARP-12 and PARP-14. In PARP-6 and PARP-8
the Glu residue appears at a different position and could be
replaced by an Asp in PARP-15, but neither the Glu nor the Asp
residues are present in PARP-7, PARP-9, PARP-10, PARP-
13 and PARP-16. For these later PARPs, no other consensus
residue appears to show up with the exception of a Pro residue
at position 213, which is observed in eleven of them (the three
hPARP-5 and PARP-7, PARP-9 to PARP-15). The absence of
the Glu catalytic residue may have an impact in limiting the
elongation activity to the synthesis of mono-ADP-ribose. The
majority of the products detected in vivo by immunological
staining (using polyclonal antibodies directed against ADP-
ribose polymers) appear to be shorter polymers.(39) The
biological importance of these PARPs lacking the Glu catalytic
residue should therefore not be neglected despite their
possibly reduced enzyme activity.
The dendrogram (Fig. 1) based on the alignment shows the
relationship between the various PARPs. Three groups can be
distinguished: the PARP-1 group containing PARP-2, PARP-3,
PARP-4, PARP-6, PARP-8 and PARP-16, the Tankyrase group
(PARP-5a, b, c) and the last group with the members PARP-7,
and PARP-9 to PARP-15. Thus, despite its comparatively short
length, the PARP motif contains sufficient variation to allow
phylogenetic discrimination. Remarkably, the catalytic domain
seems also to follow the presence of specific domains such
as the macroH2A and the WWE domains that are distributed
within two separate subgroups: PARP-9, PARP-10, PARP-14
and PARP-15 and PARP-7, PARP-11, PARP-12, and PARP-
13, respectively. Table 1 summarizes the main features of the
genes and gene products forming the PARP family.
PARPs and the mitotic apparatus
Mitosis is the most dramatic and potentially dangerous event in
the cell cycle as sister chromatids are irreversibly segregated
in daughter cells. It is therefore not surprising to find several
PARPs associated with centrosomes, telomeres, centromeres
 Review articles

Table 1. The table summarizes the main features of the PARP family sequences and gives their corresponding
GenBank identifier
Proposed Expression
nomenclature MW Aa Ip Accession# Chromosome % of PARP-1 % ident. % simil.

hPARP-1 113083 1014 9,3 XM_037275.1 1q41-42 100,0 100 100

hPARP-2 64819 570 8,6 Q9UGN5 14q11.2 14,9 46 69
hPARP-3 60088 533 6,7 XM_052494 3p21 16,6 39 61
hPARP-4 (VPARP) 192672 1724 5,3 AF158255 13q11 38,3 34 54
hPARP-5a (tankyrase1) 142011 1327 7,0 NM_003747.1 8p23.1 6,5 38 52
hPARP-5b (tankyrase2) 126917 1166 7,2 AF309033.1 10q23.03 34 54
hPARP-5c (tankyrase3) 29552 261 8,2 AK023746.1 38 54
hPARP-6 71144 630 8,4 AK022547 15q22.3 34,1 29 51
hPARP-7 (TiPARP) 78926 680 6,4 AL080156.1 3q25.31 24,4 26 50
hPARP-8 55824 501 9,5 XM_018395.2 5q11.2 9,9 25 48
hPARP-9 (Bal) 96282 854 7,9 NM_031458.1 3q13-q21 23,8 27 57
hPARP-10 109327 1020 4,6 NM_032789.1 8q24.3 35,8 28 47
hPARP-11 38738 331 7,8 AF263540 12p13.3 2,4 35 58
hPARP-12 79063 701 8,6 XM_034821.1 7q34 23,9 33 54
hPARP-14 170600 1518 8,4 XM_291055 3q21.1 31,0 30 47
HparP-15 111765 989 8,0 XM_093336 3q21.1 92,2 22 41
HparP-16 36382 322 9,4 XM_113792 15q22.2 13,6 21 32

For each sequence, the Molecular Weight (MW), the Amino acid length (Aa), and the isoelectric point (Ip) of the protein are indicated. The chomosomal
localization of the corresponding gene is based on build 33 of the NCBI human genetic map. The cDNA expression of the various PARPs is the % relative to that
of PARP-1 cDNA expression, it was established by searching the NCBI est_human database with BLAST using the corresponding cDNA. The amino acid
conservation (% of identity and % of similarity) is given only for the PARP catalytic domain relative to that of PARP-1.

and microtubules (Fig. 3). Recent studies on syncytial
Drosophila embryos demonstrated that centrosome inactiva-
tion is part of the mitotic response to DNA lesions that blocks
chromosome segregation and prevents proliferation of cells
with genomic instability.(40) This response appears to require
the localization of the checkpoint kinase Chk2 to the centro-
some. It remains to be determined whether higher eukaryotes
show a comparable centrosomal response to impaired DNA
integrity and if PARP-1 and/or PARP-3 play a role in this
process.
Remarkably, a number of recently identified partners of
PARP-1 and PARP-2 such as CENP-A, CENP-B, Bub 3(41–43)
and Aurora B (L. Monaco, R. Loury, J. Ménissier-de Murcia, M.
Parvinen, G. de M. and P. Sassone-Corsi, unpublished data)
are clearly involved in the organization of pericentric chromatin
and in the mechanics of cell division. In response to DNA
damage during mitosis, poly(ADP-ribosyl)ation of Aurora B by
PARP-1 and/or PARP-2 has been shown to negatively
regulate its kinase activity on Ser10 of Histone H3, thus halting
metaphase progression (L. Monaco, R. Loury, J. Ménissier-de
Murcia, M. Parvinen, G. de M. and P. Sassone-Corsi,
unpublished data). In addition, the presence in the midbody
of PARP-1 and PAR following DNA damage (C.S. and G. de M.
unpublished results) suggest a possible function in a detec-
tion–signaling pathway aimed at monitoring the eventual
presence of broken DNA that originates from tension forces

Figure 3. The PARP superfamily and the cell

division. Subcellular localization of some of the
PARP family members to the mitotic apparatus
(see text for details).

BioEssays 26.8 887

Review articles
between two daughter cells experiencing unbalanced chro-
mosome segregation. Therefore, PARP-1 displays most of the
characteristic behavior of a chromosomal passenger that
associates with kinetochores of cells prepared for nuclear
division and later on localizes to the bundled filaments forming
the central spindle and finally the contractile ring. The various
localizations of PARP-1 during the metaphase–anaphase
transition as well as the anaphase–telophase transition
strongly suggest a possible role in maintaining the genome
stability and segregation fidelity during cell division by con-
troling the cell cycle progression at various stages in response
to DNA damage.
PARP-2 also localizes to centromeres during prometa-
phase and shares with PARP-1 the same partners in this
compartment: CENP-A, CENP-B and Bub3.(43) Interestingly,
the disruption of the mouse parp-2 gene lead to sensitization to
ionizing radiation and monoalkylating agents(15) associated
with preferential centromeric breaks, a prominent G2/M
accumulation, acquisition of 8N DNA content and a significant
level of anaphase bridges. The elevated rate of chromosome
mis-segregation is reminiscent of kinetochore defects that
take place in PARP-2-deficient mice irradiated with low doses
of g-rays. In summary, these data suggest that the presence of
several PARPs at kinetochores, makes these key regions of
our chromosome-sensitive integrators of DNA strand breaks,
capable of instantly interrupting the process of microtubule
capture to prevent mitotic errors.
Double mutant mice (parp-1
/
parp-2
/
) are not viable
and die at the onset of gastrulation demonstrating that DNA-
dependent poly(ADP-ribosyl)ation is essential during early
embryogenesis. Surprisingly, specific female embryonic leth-
ality is observed in parp-1þ/
parp-2
/
(but not in parp-1
/

parp-2þ/
mice) accompanied by a specific instability of
the X chromosome.(15) A defective segregation of the
X chromosome, already known as a lagging chromosome,
can be evoked. The already known lagging character of
the inactivated X chromosome(44) could be associated with the
fact that, as a silenced region of the genome, it may be less
frequently repaired than actively transcribed portions and
therefore needs to be repaired during DNA replication to be
faithfully transmitted. Depending on the extent of DNA
damage, the inactivated X chromosome might not be captured
in time by the microtubules, this delay is most probably
accentuated in the parp-2
/
context, thus increasing its
instability.
The dual role of poly(ADP-ribose)
synthesis in response to DNA strand breaks:
chromatin loosening and XRCC1 recruitment
Changes in chromatin structure resulting from DNA breaks are
probably the earliest events in the cellular response to DNA
damage. Poly(ADP-ribosyl)ation of histones and nuclear
proteins as well as phosphorylation of histone H2AX on
888 BioEssays 26.8
Ser139(45) are two epigenetic marks induced by DNA strand
breaks that contribute to the histone code in damaged
chromatin. DNA-damage-induced poly(ADP-ribosyl)ation of
H1 and H2B contributes to the relaxation of the 30 nm
chromatin fibre in vitro (Fig. 4) and we have proposed a long
time ago that this immediate conformational change of the
nucleosomal superstructure may trigger the access of repair
enzymes to the lesion in vivo.(12) Non-covalent interactions
between histone tails and PAR also take place, further
decreasing nucleosomal stability.(46)
The role of PARP-1 during the repair of single-strand
breaks in mammalian cells is now better understood, not only
in vitro where PARP-1 activity facilitates the whole process,
but also in living cells where PAR synthesis at sites of DNA
lesions plays an essential role in the recruitment kinetics of a
key player: XRCC1.(47,48) This remarkable property observed
by Okano et al. is illustrated in Fig. 4, but with a different
experimental setting. A robust PAR synthesis is immediately
detected in proliferating cells irradiated with a laser microbeam
at 337 nm in the presence of Hoechst. The absence of dynamic
recruitment of XRCC1 in PARP-1-deficient cells could thus
explain the important delay in strand-break rejoining that, in
turn, causes a severe DNA repair defect.(49)
PARP-1 activity also occurs during the repair of damaged
bases by BER. Activation of polymer synthesis likely follows
the incision step of the sugar-phosphate backbone by DNA
glycosylase-AP lyases or APE-1. We have shown that the
polymerization step of the Long Patch Repair (LPR) pathway
was mainly affected in PARP-1-deficient cells.(50) Moreover,
PARP-1 is associated with DNA pol-b(50,51) and efficiently
binds to the repair intermediates containing a flap50 -abasic site
that are formed before sub-pathway choice, leading to either
Short Patch Repair (SPR) or LPR.(51,52) The ligation step is
also affected by the presence of PARP-1 since it was demo-
nstrated that a direct physical interaction between PARP-1
and DNA ligase III occurs via the region of amino acids
immediately adjacent to the N-terminal zinc finger of DNA
ligase III.(53) It was further demonstrated that DNA ligase III
also binds directly to PAR and to poly(ADP-ribosyl)ated
PARP-1, leading to an increase in DNA joining. This provides
a mechanism for the recruitment of the DNA ligase III–XRCC1
complex at DNA breaks. Therefore the presence of PARP-1
and/or PAR throughout the BER/SSBR process, in interaction
with XRCC1 and DNA Ligase III, which are not found in lower
eukaryotes, suggests a unique role in maintaining genome
stability in complex organisms.
It is not known whether the phosphorylation of histone
H2AX on Ser139 also contributes to chromatin accessibility
after damage. It seems rather to induce the recruitment of
double-strand-break repair factors and checkpoint control
proteins at damaged foci providing a means of ordering the
molecular events ensuing from DSB detection.(54,55) In
fact, the epigenetic mark put to histone H2AX in response to
 Review articles

Figure 4. The multiple functions of PAR in the cellular response to DNA damage. In response to DNA damage, DNA-damage-dependent
PARPs detect(75) and signal interruptions in the double helix. DNA-break-induced poly(ADP-ribosyl)ation of histone H1 and PARPs leading,
in a few seconds, to the relaxation of chromatin structure(12,76) and simultaneously to the recruitment of XRCC1(47) and SSBR enzymes to
the damage site, thus increasing the repair kinetics. The native chromatin structure (30 nm fibre) is finally restored after degradation
of PAR by PARG. Under certain physiological conditions, the persistence of free polymers (PAR) may have a stress-signaling function
(see also Fig. 5).

DNA-strand break can occur independently of PAR synthesis
or of the PARP-1 and PARP-2 status.(56) Thus, co-localization
but no connection apparently occurs between these two post-
translational modifications acting on chromatin structure in
response to DNA breakage. It is worth recalling that the
simultaneous loss of both modifications in the double mutation
(parp-1
/
; atm
/
)(57) leads to early embryonic lethality
suggesting the absolute necessity of maintaining at least one
of these two important pathways, especially during early
embryonic development.
The case of D. melanogaster
Recent studies have shown that PARP-1 and/or its enzymatic
activity are directly involved in transcription regulation in
Drosophila where it is possible to visualize the activity of genes
at specific loci on polytene chromosomes.(58) In addition,
Drosophila is a suitable model to study PARP biology because
it contains just two PARP genes, a PARP-1 like gene and
tankyrase. In the fruit fly, the transcriptional response is rapid,
producing many mRNA transcripts, fading shortly after the
inducing signal disappears. This rapid transcriptional re-
sponse is clearly visible under the microscope as ‘‘puffs’’ in
the giant polytene chromosomes of the salivary glands. Within
minutes of a heat-shock stimulus, the chromosomal sites of
heat-shock genes become decondensed, swell to many times
their original diameter, and accumulate many new mRNA
transcripts. The same puffs occur at chromosomal sites of
hormone-responsive genes in response to increased pro-
duction of steroid hormone in larvae approaching pupation.
Tulin and Spradling(58) showed that PARP-1 is crucial for puff
formation in Drosophila polytene chromosomes. PARP-1 is
widely distributed on these chromosomes and is normally
inactive. However, after a heat-shock stimulus, the protein
accumulates rapidly at heat-shock loci where an intense PAR
synthesis activity occurs. A similar PARP accumulation and
activation is noted in ecdysone-induced puffs in larvae before
pupation. Another target for PARP-1 is the nucleolus, where
rRNA genes are the sites of the major transcriptional activity of
the cells even though they are surrounded by condensed
heterochromatin that usually inhibits transcription. Transcrip-
tion and puffs formation is largely reduced by PARP-1
mutations or by the PARP inhibitor 3-aminobenzamide. The
study reveals a strong correlation between PAR accumulation
and puffing. In addition, the authors found that PARP mutant
Drosophila are extremely sensitive to bacterial infections
due to a defective activation of genes involved in the control
of the immune response, similar to NF-kB homologues
rejoining the phenotypes observed for the PARP-1 knockout

BioEssays 26.8 889

Review articles
mice.(59) These observations argue that PARP’s role in NF-kB-
dependent immune response gene expression has been
conserved during evolution. PARP-1 may act on the chromatin
organization of NF-kB target loci. Signals other than DNA
lesions, including steroid hormones, stress, and infection, may
activate PARP molecules at specific chromosome sites. After
modification with PAR, local chromatin proteins, transcription
factors, and PARP-1 molecules themselves are proposed to
dissociate from DNA and/or from pre-existing protein com-
plexes, and to complex with nearby branched ADP-ribose
polymers. Consequently, the modified chromosome domain
may be transcribed and its chromatin constituents modified.
Later on, due to the action of the poly (ADP-ribose) glyco-
hydrolase (PARG) that cleaves PAR,(60) the chromatin would
reform to return to the initial state.
Many questions still need to be answered, among them is
the understanding of how PARP-1 is recruited to target genes
and activated within normal, undamaged chromatin. Given
that topoisomerase I is a necessary player in transcription and
a partner of PARP-1, it is quite possible that, under a variety of
circumstances, notably when transcription is particularly
active as in puffs or in complex DNA sequence regions,
abortive topoI-SSB may arise leaving transient 50 DNA ends
that particularly stimulate PARP-1.(61) A spontaneous PARP-1
activation, as the one observed in Drosophila puffs, could well
be the result of transient topoisomerase I pauses. Recipro-
cally, it has been recently shown that PAR formation at stalled
topoisomerases I activates DNA break resealing.(62)
Could these mechanisms described in the Drosophila be
generalized to mammalians? If so what could be the role of
the homologues of PARP-1, like PARP-2, for which some
redundant functions have already been described?
The dark side of PARP-1: from the inflammatory
response to AIF-mediated cell death
The generation, by homologous recombination, of three
independent deficient mouse models have fully confirmed
the caretaker function of PARP-1 in mammalian cells
challenged by various genotoxins that directly or indirectly
break the DNA.(63–65) Unexpectedly, the knockout strategy
revealed the instrumental role of PARP-1 in cell death in
various acute and chronic inflammatory disorders (see for
review(8)). There has been increasing evidence that PARP-1
is, in fact, involved in the inflammatory response at two
different levels: (i) activation of NF-kB and synthesis of
proinflammatory factors(66) and (ii) increasing the sensitivity
of cells (particularly endothelial cells) to oxygen radicals
produced during inflammation with consequences on DNA
damage, energy depletion and cell death. More recently,
Dawson and colleagues provided evidence that PARP-1
activity participates in the mechanism leading to cell death
through the release of a mitochondrial proapoptotic protein
called apoptosis-inducing factor (AIF).(67) AIF is a flavoprotein
890 BioEssays 26.8
normally confined to the mitochondrial intermembrane space
in healthy cells. On induction of cell death by several
apoptogenic agents, AIF translocates to the nucleus and
induces chromatin condensation and large-scale (50 kbp)
DNA fragmentation in a new form of caspase-independent
apoptosis termed chromatinolysis.(68) Subsequent to its
migration from mitochondria, AIF also triggers the release of
mitochondrial cytochrome c and caspase activation. However,
blockade of caspase activation does not abrogate AIF-
dependent apoptotic cell death. How can PARP-1 induce
AIF translocation from the mitochondria to the nucleus?
The authors showed that the activity of PARP-1 is absolutely
required for this process. The treatment of fibroblasts or
cortical neurons with very high doses of DNA-damaging
agents such as MNNG, NMDA or H2O2, induces cell death
through the translocation of AIF but the translocation is
inhibited in the presence of PARP inhibitor 3,4-dihydro-5-
[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ) or in
PARP-1-deficient cells.(67) AIF-neutralizing antibodies could
block apoptosis but not caspases inhibitors despite the
presence of features indicative of caspase-dependent cell
death (such as cytochrome c release, caspase activation and
PARP-1 cleavage).(69) In addition, the overexpression of Bcl-
2, one of the most powerful antiapoptotic proteins identified so
far, delayed but did not prevent PARP-1-dependent apoptosis.
Interestingly, it has been demonstrated that AIF is involved
in neuronal as well as myocardial cell death by ischemia-
reperfusion(70,71) in which PARP-1 plays a critical role. On the
one hand, a very rapid signaling by nuclear PARP-1 to the
mitochondria seems to cause AIF translocation. On the other
hand, PAR accumulation in the central nervous system was
recently demonstrated in Drosophila mutants in which the
gene encoding the PAR degrading enzyme, poly(ADP-ribose)
glycohydrolase (PARG), was disrupted leading to progressive
neurodegeneration, reduced locomotion and short life-
span.(72) The key question that remains is which signal emitted
by the nucleus informs the mitochondria to release AIF?
Whatever the answer, AIF, as an essential downstream ef-
fector of PARP-1-mediated cell death, appears to be a future
therapeutic target in a number of inflammatory disorders.
Conclusions
Most of the published data on PARP-1 and PARP-2 integrate
the unique property of these two DNA-dependent enzymes to
detect DNA interruptions acting as potent enzymatic coacti-
vators during various physiological or pathological processes.
They clearly participate in the first line of defense of the
genome as efficient DNA-break sensors and signaling
molecules as a part of a survival program. In contradiction,
the response of a cell in which AIF has migrated from the
mitochondria to the nucleus is that the same enzymes now
participate actively in cell death (Fig. 5). These two facets of
PARP activity will undeniably continue to fascinate PARP
 Review articles

Figure 5. The two facets of PARP activation. In replicating cells, limited damage to DNA induces PARP activity that allows the activation of
DNA repair pathways through the recruitment of DNA repair factors (i.e. XRCC1). The decision for the cell to engage the apoptotic pathway
after genotoxic stress takes place downstream of p53 activation, and the molecular determinants that switch between DNA repair and cell-
cycle arrest or apoptosis are not yet fully understood. As a consequence of the apoptosis option, PARPs are cleaved by caspases and are
thus inactivated to avoid futile repair. PARP inhibitors (red route) potentiate the cytotoxicity of DNA damaging agents. In post-mitotic cells,
reactive oxygen species (ROS) damage DNA, which in turn activates PARP and triggers AIF translocation to the nucleus.(67) The resulting
chromatinolysis exacerbates PARP activity and NAD depletion. The instrumentalization of PARP under these pathological conditions may
be responsible for the inflammatory status of the tissue/organ. PARP inhibitors are known to prevent both AIF translocation and
inflammatory injury.

aficionados for a while. PARP involvement in both cell survival
and cell death gives rise to an intense industrial effort to
develop potent chemical inhibitors that, at the preclinical
phase, are now able to cause a complete regression of xeno-
graft tumors.(73) Among the other members of the PARP
family, the Tankyrase group, which is involved in telomere
functioning, and the MacroPARP group, whose function could
be associated with chromatin repression, may well constitute
exciting fields of development for basic and applied science.
Acknowledgments
We thank all the members of the de Murcia laboratory and O.
Poch for fruitful discussions. We also thank Dr. S. Natarajan-
Amé for careful reading of the manuscript. We are grateful to
Didier Hentsch (IGBMC, Illkirch), Jean-Christophe Laval
(Leica Microsystems, France) for their help with the laser
microbeam microscope. We thank Dr. Sugimura for the 10H
anti-poly(ADP-ribose) antibody.
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