Tài liệu Báo cáo khoa học: Poly(ADP-ribose) The most elaborate metabolite of NAD+ Alexander Burkle pptx

MINIREVIEW
Poly(ADP-ribose)
The most elaborate metabolite of NAD
+
Alexander Bu
¨
rkle
Department of Biology, University of Konstanz, Germany
Introduction
The life cycle of poly(ADP-ribose)
NAD
+
⁄ NADH is among the most versatile biomole-
cules, as it can be used not only as a coenzyme for a
large number of oxidoreduction reactions, but in its
oxidized version can also serve as substrate for several
different of ADP-ribosyl transfer reactions, which are
the overarching theme of this minireview series. The
covalent transfer onto glutamic acid, aspartic acid or
lysine residues of target proteins (‘acceptors’), followed
by successive transfer reactions onto the protein–
mono(ADP-ribosyl) adduct, and subsequently onto the
emerging chain of several covalently linked ADP-ribo-
syl residues is the basis of the formation of poly(ADP-
ribose), which can be regarded the cell’s most elaborate
metabolite of NAD
+
[1]. ADP-ribose chains may com-
prise up to 200 ADP-ribose units, coupled via unique
ribose (1¢¢fi2¢) ribose phosphate-phosphate linkages
and display several branching points resulting from the

formation of ribose (1¢¢¢fi2¢¢) ribose linkages (Fig. 1).
Poly(ADP-ribosyl)ation occurs in multicellular organ-
isms including plants and some lower unicellular eu-
karyotes, but is absent in prokaryotes and yeast.
Poly(ADP-ribosyl)ation is catalysed by the family of
poly(ADP-ribose) polymerases (PARPs; Fig. 2), enco-
ded in human cells by a set of 18 different genes [2].
Keywords
PARP; tankyrase; poly(ADP-ribose); DNA
damage; DNA repair; genomic instability;
centrosome; centromere; telomeres; mitotic
spindle
Correspondence
A. Bu
¨
rkle, Department of Biology,
Box X911, University of Konstanz,
D-78457 Konstanz, Germany
Tel: +49 7531 884035
Fax: +49 7531 884033
E-mail:
Website: logie.
uni-konstanz.de/
(Received 5 May 2005, accepted 14 July
2005)
doi:10.1111/j.1742-4658.2005.04864.x
One of the most drastic post-translational modification of proteins in eu-
karyotic cells is poly(ADP-ribosyl)ation, catalysed by a family enzymes
termed poly(ADP-ribose) polymerases (PARPs). In the human genome, 18
different genes have been identified that all encode PARP family members.

Poly(ADP-ribose) metabolism plays a role in a wide range of biological
structures and processes, including DNA repair and maintenance of
genomic stability, transcriptional regulation, centromere function and mito-
tic spindle formation, centrosomal function, structure and function of vault
particles, telomere dynamics, trafficking of endosomal vesicles, apoptosis
and necrosis. In this article, the most recent advances in this rapidly grow-
ing field are summarized.
Abbreviations
ANK, ankyrin; BER, base excision repair; BRCA1, breast cancer 1 protein; DBD, DNA-binding domain; HPS, His-Pro-Ser-rich; IRAP, insulin-
responsive amino peptidase; MVP, major vault protein; NuMa, nuclear mitotic apparatus protein; PARG, poly(ADP-ribose) glycohydrolase;
PARP, poly(ADP-ribose) polymerase; RNP, ribonucleoprotein particle; Sir2, silent information regulator 2; TCDD, 2,3,7,8-tetrachlorodibenzo-
p-dioxin; TRF, telomeric-repeat binding factor.
4576 FEBS Journal 272 (2005) 4576–4589 ª 2005 FEBS
Apart from the covalent modification of acceptor
proteins, poly(ADP-ribose) has also been shown to
interact noncovalently, yet specifically, with a wide
range of proteins [3]. This interaction is mediated
through a sequence motif displaying a conserved pat-
tern, and it is interesting to note that DNA damage
checkpoint proteins such as p53 and p21 possess such
poly(ADP–ribose) interaction domains. DNA methyl-
transferase-1 (DNMT1) is also able to bind long and
branched ADP-ribose polymers in a noncovalent fash-
ion, which leads to inhibition of DNMT1 activity [4].
Taken together, poly(ADP-ribose) can also affect the
function of proteins that are not direct modification
targets.
Enzymes involved in poly(ADP-ribose)
metabolism
PARP-1

The prototypic enzyme of the PARP family is poly
(ADP-ribose) polymerase-1 (PARP-1; EC 2.4.2.30;
Table 1). Its discovery was made four decades ago by
Chambon, Weill and Mandel [5] and marked the birth
of a most interesting and intriguing field originally
occupied by biochemists, but later on also attracting
radiobiologists, toxicologists, geneticists, molecular
biologists, pharmacologists, cell biologists and experts
from other biological disciplines.
Nicotinamide
NAD
+
P
AR
P
AR
n NAD
+
n Nicotinamide + n H
+
Rib
O
O
PO
O
P
O
Rib O
-
O

-
N
N
NH
2
N
N
Acceptor
protein
CH
2
C
O
O
-
Rib
N
N
NH
2
N
N
O
O
O
-
PO
O
O
-

P
O
Rib
Rib
N
N
NH
2
N
N
O
O
O
-
PO
O
O
-
P
O
Ri
b
Rib
Rib
N
N
NH
2
N
N

O
O
O
-
PO
O
O
-
P
O
Rib
Rib
N
N
NH
2
N
N
O
O
O
-
PO
O
O
-
P
O
Rib
Rib

N
N
NH
2
N
N
O
O
O
-
PO
O
O
-
P
O
Rib
Rib
N
+
H
2
NCO
N
H
2
NCO
+ H
+
Rib

N
N
NH
2
N
N
O
O
O
-
PO
O
O
-
P
O
Rib
+
CH
2
C
O
O
CH
2
C
O
O
Acceptor
protein

Acceptor
protein
Fig. 1. Poly(ADP-ribose) structure.
Poly(ADP-ribose) polymerases (PARPs)
cleave the glycosidic bond of NAD
+
between nicotinamide and ribose followed
by the covalent modification of mainly glu-
tamate residues of acceptor proteins with
an ADP-ribosyl unit. PARPs also catalyse
an adduct elongation, giving rise to linear
polymers with chain lengths of up to about
200 ADP-ribosyl units, characterized by
their unique ribose (1¢¢fi2¢) ribose phos-
phate-phosphate backbone. At least some
of the PARP family members also catalyse
a branching reaction by creating ribose
(1¢¢¢fi2¢¢) ribose linkages. The sites of
hydrolysis catalysed by poly(ADP-ribose)
glycohydrolase (PARG), the major poly(ADP-
ribose)-degrading enzyme, are indicated
by arrows.
A. Bu
¨
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FEBS Journal 272 (2005) 4576–4589 ª 2005 FEBS 4577
PARP-1 is a highly conserved and abundant nuclear
protein, which is catalytically active as a dimer and is
the major acceptor protein in intact cells, via the
so-called automodification reaction. A range of other

nuclear proteins can also serve as acceptor proteins
(see below). PARP-1 displays a characteristic three-
domain structure (Fig. 2), which can be further broken
down into modules A–F [6]. The N-terminal 42 kDa
DNA-binding domain (DBD) also comprises the pro-
tein’s nuclear localization signal and is adjacent to a
central 16 kDa automodification domain. The 55 kDa
catalytic domain, which includes the active site, is
located at the C-terminus.
The DBD of PARP-1 binds to single- or double-
strand breaks with high affinity via two zinc fingers
but has also been reported to be involved in protein–
protein interactions. The first zinc finger is essential for
PARP-1 activation by DNA strand breaks, whereas
the second is essential for PARP-1 activation by DNA
single-strand breaks but not double-strand breaks. In
the absence of DNA breaks, PARP-1 displays a very
low basal enzyme activity. The molecular mechanism
linking DNA binding to catalytic activation is
unknown. Automodification of PARP-1 was reported
to be mostly induced by single-strand breaks, whereas
histone H1 seems to be modified preferentially when
PARP-1 binds to double-strand breaks [7]. Following
up earlier reports [8–10], several groups have provided
recent evidence for alternative activation mechanisms
for PARP-1, independent of DNA strand breakage.
PARP-1 was reported to recognise distortions in the
Table 1. Human enzymes involved in poly(ADP-ribose) formation or degradation and their genes.
Official gene
symbol Name [aliases]

Chromosomal
location
Gene
ID Protein size
PARP1 Poly(ADP-ribose) polymerase family, member 1
[PARP-1, ADPRT, ADPRT1, PARP, PPOL, pADPRT-1]
1q41-q42 142 1014 aa (113 kDa)
PARP2 Poly(ADP-ribose) polymerase family, member 2
[PARP-2, NAD
+
poly(ADP-ribose) polymerase-2]
14q11.2-q12 10038 583 aa (66 kDa)
PARP3 Poly(ADP-ribose) polymerase family, member 3
[PARP-3, NAD
+
poly(ADP-ribose) polymerase-3]
3p22.2-p21.1 10039 532 aa (60 kDa);
splice variant
539 aa (60.8 kDa)
PARP4 Poly(ADP-ribose) polymerase family, member 4
[PARP4, ADPRTL1, PARPL, PH5P, VAULT3, VPARP, p193]
13q11 143 1724 aa (192.8 kDa)
TNKS Tankyrase, TRF1-interacting ankyrin-related ADP-ribose
polymerase [PARP-5a, PARP5A, PARPL, TIN1, TINF1, TNKS1]
8p23.1 8658 1327 aa (142 kDa)
TNKS2 Tankyrase-2, TRF1-interacting ankyrin-related
ADP-ribose polymerase 2 [PARP-5b, PARP-5c,
PARP5B, PARP5C, TANK2, TNKL]
10q23.3 80351 1166 aa (126.9 kDa)
TIPARP TCDD-inducible poly(ADP-ribose) polymerase

[PARP-7]
3q25.31 25976 657 aa (75 kDa)
PARP10 Poly (ADP-ribose) polymerase family, member 10
[PARP-10]
8q24.3 84875 1025 aa (150 kDa)
PARG Poly(ADP-ribose) glycohydrolase
[PARG]
10q11.23 8505 Three splice variants
976 aa (111 kDa);
893 aa (102 kDa);
866 aa (99 kDa)
PARP-1
PARP-2
PARP-3
PARP-4
(VPARP)
Tankyrase
(PARP-5a)
Tankyrase-2
(PARP-5b)
N
BRCT
NLSZFI ZFII
Module
A B C E DF
DNA-binding domain (DBD)
NAD
+
-binding domain
Automodification domain

Active site
DBD
NLS
BRCT
SA
24 ankyrin
HPS
SA
24 ankyrin
VIT TM VWA SH3 MVP-BD
C
Fig. 2. Structural organization of some PARP protein family mem-
bers. BRCT, BRCA1 C-terminus; DBD, DNA-binding domain; HPS,
His-Pro-Ser-rich domain; MVP-BD major vault binding domain; NLS,
nuclear localization signal; SAM, sterile a-module; SH3, src homo-
logy region; TM, transmembrane domain; VIT, vault protein inter-
alpha-trypsin domain; VWA, von Willebrand factor type A domain;
ZF; zinc finger.
Poly(ADP-ribosyl)ation A. Bu
¨
rkle
4578 FEBS Journal 272 (2005) 4576–4589 ª 2005 FEBS
DNA helical backbone and to bind to three- and four-
way junctions as well as to stably unpaired regions in
double-stranded DNA [11]. Such PARP-1 interactions
with non-B DNA structures led to catalytic activation
of the enzyme in the absence of free DNA ends. DNA
hairpins, cruciforms and stably unpaired regions are
all effective coactivators of PARP-1 automodification
and poly(ADP-ribosyl)ation of histone H1. These data

suggest a link between PARP-1 binding to non-B
DNA structures in the genome and its function in the
dynamics of local modulation of chromatin structure
in normal cellular physiology (see below). Binding to
and activation by unbroken DNA in the context of
chromatin has also been reported to play a crucial role
in the NAD
+
-dependent modulation of chromatin
structure and transcriptional regulation, mediated by
PARP-1 [12]. Along the same lines it was suggested
that fast and transient decondensation of chromatin
structure by poly(ADP-ribosyl)ation occurring in Aply-
sia neurones in the absence of DNA strand breaks
enables the transcriptional regulation needed to form
long-term memory in this organism [13].
The automodification domain of PARP-1 is rich in
glutamic acid residues, consistent with the fact that
poly(ADP-ribosy)lation occurs on such residues. This
domain also comprises a breast cancer 1 protein
(BRCA1) C-terminus (BRCT) motif that is present in
many DNA damage repair and cell-cycle checkpoint
proteins.
The C-terminal 55 kDa catalytic domain contains the
residues essential for NAD
+
-binding, ADP-ribosyl
transfer and branching reactions. The crystal structure
of the C-terminal catalytic fragment revealed a striking
homology with bacterial toxins that act as mono(ADP-

ribosyl) transferases [14].
While PARP-1 is constitutively expressed, its charac-
teristic ability of being activated by DNA strand
breaks makes poly(ADP-ribosyl)ation an immediate
and drastic cellular response to DNA damage as
induced by ionizing radiation, alkylating agents and
oxidants. In the absence of DNA single and double
strand breaks, poly(ADP-ribosyl)ation seems to be a
very rare event in live cells, but it can increase over
100-fold upon DNA damage [15]. Under these condi-
tions about 90% of poly(ADP-ribose) is synthesized
by PARP-1 [16].
Among many identified interaction partners of
PARP-1 are also other members of the PARP-family,
such as PARP-2 [17–19] and PARP-3 [20]. As men-
tioned above, the most prominent target protein
(acceptor) of this poly(ADP-ribosyl)ation reaction is
PARP-1 itself but many other acceptor proteins have
been described, including p53 [21], both subunits of
NF-jB [22], histones, DNA-topoisomerases and the
catalytic subunit of DNA-dependent protein kinase
(DNA-PK
cs
). Due to the high negative charge of the
polymer, this modification significantly alters the phys-
ical and biochemical properties of the modified pro-
teins, such as their DNA-binding affinity, and it is
likely that such alteration will have a regulatory func-
tion concerning the interaction with other proteins
[23].

Three different PARP-1 knockout mouse models
(Parp1
– ⁄ –
) have been created independently [24–26],
lacking the PARP-1 protein. Parp1
– ⁄ –
mice are viable
and fertile, but show hypersensitivity to alkylation
treatment or ionizing radiation and loss of genomic
stability. In stark contrast, however, they display pro-
tection against various pathophysiological phenomena,
such as lipopolysaccharide-induced septic shock or
streptozotocin-induced diabetes [27].
PARP-2
Apart from PARP-1, PARP-2 is the only other PARP
isoform known to be activated by DNA strand breaks
[28] (Table 1; Fig. 2). This enzyme displays automodifi-
cation properties similar to PARP-1. Its DBD, how-
ever, is different from that of PARP-1, consisting of
only 64 amino acids and lacking any obvious DNA-
binding motif. The crystal structure of the catalytic
fragment of murine PARP-2 has recently been solved,
thus providing a basis for the development of isoform-
specific inhibitors by rational drug design [29]. Despite
major structural differences between PARP-1 and
PARP-2, including size and the absence of zinc fingers
or the BRCT motif, they are both targeted to the nuc-
leus, and they bind to and become activated by DNase
I-treated DNA. Both PARP-2 and PARP-1 can homo-
and heterodimerise, and both are involved in the base

excision repair (BER) pathway where they form a
complex with X-ray cross-complementing factor 1
(XRCC1) [18]. Parp1
– ⁄ –
⁄ Parp2
– ⁄ –
double mutant mice
are nonviable and die at the onset of gastrulation,
highlighting that the expression of both PARP-1
and PARP-2 and ⁄ or DNA strand break-dependent
poly(ADP-ribosyl)ation is essential during early embryo-
genesis.
PARP-3
The protein domain structure of PARP-3 is very
similar to PARP-2, featuring a small DNA-binding
domain consisting of only 54 amino acids and compri-
sing a centrosome-targeting motif [20]. Overexpression
of PARP-3 or its N-terminal domain in HeLa cells
A. Bu
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FEBS Journal 272 (2005) 4576–4589 ª 2005 FEBS 4579
interfered with the G
1
⁄ S cell cycle transition. PARP-3
catalyses the synthesis of poly(ADP-ribose) in vitro
and in purified centrosome preparations, and forms a
stable complex with PARP-1, in agreement with other
reports of PARP-1 localization at the centrosome
[30,31].

PARP-4
Vault particles are cytoplasmic ribonucleoprotein parti-
cles (RNPs) composed of several small untranslated
RNA molecules and three proteins of 100, 193 and
240 kDa. With a total mass of 13 MDa, vaults are the
largest RNP complexes found in the cytoplasm of
mammalian cells. The 193 kDa vault protein was iden-
tified as a novel PARP [32,33] and is now termed
PARP-4 (Table 1, Fig. 2). PARP-4 poly(ADP-ribo-
syl)ates the p100 subunit (major vault protein; MVP)
within the vault particle and to a lesser extent itself.
The N-terminal region of PARP-4 contains a BRCT
domain similar to the automodification domain of
PARP-1, suggestive of a related function.
Tankyrase
This protein was initially identified through its inter-
action with the telomeric-repeat binding factor 1
(TRF1), which is another negative regulator of telo-
mere length [34]. The N-terminus of tankyrase contains
a so-called His-Pro-Ser-rich (HPS) domain consisting
of stretches of consecutive histidine, proline and serine
residues, followed by 24 ankyrin (ANK) repeats, which
is a structural feature only found in tankyrase and
tankyrase-2 (see below) within the known members of
the PARP family. Adjacent to the ANK domain is
another protein interaction motif, the sterile alpha-
module. The C-terminus of tankyrase displays homo-
logy to the PARP-1 catalytic region. Consistent with
the absence of any DNA-binding domain, tankyrase
activity does not depend on the presence of DNA

strand breaks but seems to be regulated by the phos-
phorylation state of the protein [35]. About 10% of
cellular tankyrase protein is recruited to telomeres
through binding of its ANK domain to TRF1. Thus
the binding of TRF1 to telomeric DNA controls telo-
mere length in cis by inhibiting the action of telo-
merase at the ends of individual telomeres [36].
Tankyrase-2
This enzyme was originally described as a tumour anti-
gen that elicited antibody responses in certain tumour
patients (Table 1, Fig. 2) [37,38]. Later, tankyrase-2
was reported to interact with several other proteins
such as TRF1 [39], insulin-responsive amino peptidase
(IRAP) [40], or Grb14, an SH2 domain-containing
adaptor protein that binds to the insulin and fibroblast
growth factor receptors [41]. Despite being encoded by
a separate gene, tankyrase-2 displays a domain struc-
ture that is strikingly similar to tankyrase except for
the N-terminal HPS domain, which is missing in
tankyrase-2 (Fig. 2) [39,41]. Tankyrase and tankyrase-
2 also show a significant functional overlap: both pro-
teins possess PARP activity and poly(ADP-ribosyl)ate
some of their interaction partners (IRAP, TAB182 and
TRF1, but not TRF2) as well as themselves, whereas
tankyrase-2 displays preferential automodification
activity. Strikingly, overexpression of tankyrase-2, but
not of tankyrase, caused rapid poly(ADP-ribo-
syl)ation-dependent cell death [39]. Both tankyrases
can self-associate via the sterile alpha-module domain
to form high-molecular-mass complexes, indicative of

a function as master scaffolding molecules in organ-
izing protein complexes [42].
PARG
While there are 18 genes currently known or assumed
to encode different PARP isoforms [2] there is only a
single gene known to encode an enzyme catalysing the
hydrolysis of ADP-ribose polymers to free ADP-
ribose. This is the gene encoding poly(ADP-ribose)
glycohydrolase (PARG; EC 3.2.1.143; Table 1) [43–45].
The human PARG gene shares a 470 bp bidirectional
promoter with the gene encoding the translocase of the
inner mitochondrial membrane 23 (TIM23). Promoter
activity is several fold higher for TIM23 than for
PARG [46]. Three splice variants of the human PARG
have been described, giving rise to PARG isoforms tar-
geted either to the nucleus or to the cytoplasm [47].
Overexpression studies revealed that the largest iso-
form of PARG is targeted to the nucleus while the two
smaller isoforms show mostly cytoplasmic localization.
PARG is an enzyme that possesses both endoglyco-
sidic and exoglycosidic activity and is the only protein
known to catalyse the hydrolysis of ADP-ribose poly-
mers to free ADP-ribose (Fig. 1) [43]. Its products are
free poly(ADP-ribose) and monomeric ADP-ribose,
the latter being a potent protein-glycating carbohy-
drate capable of causing protein damage [48]. Interest-
ingly, an ADPR pyrophosphatase has been described
that converts ADPR to AMP and ribose 5-phosphate,
thus decreasing the risk of nonenzymatic protein gly-
cation [49]. As a consequence of the combined action

of PARPs and PARG, poly(ADP-ribose) undergoes a
dynamic turnover in live cells.
Poly(ADP-ribosyl)ation A. Bu
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4580 FEBS Journal 272 (2005) 4576–4589 ª 2005 FEBS
Biological functions of poly(ADP-
ribosyl)ation
DNA repair and maintenance of genomic stability
Mechanistic aspects
A plethora of studies have firmly established that DNA
damage-induced poly(ADP-ribosyl)ation contributes to
cellular recovery from cytotoxicity in proliferating cells
inflicted with low or moderate levels of DNA damage
by alkylation, oxidation or ionizing radiation. PARP-1
as well as the related protein PARP-2 are those members
of the PARP family that are responsive to DNA damage
and play important roles in DNA repair and mainten-
ance of genomic integrity, thus behaving as ‘survival fac-
tors’ [50,51]. Specifically, PARP-1 and PARP-2 have
been shown to play a crucial role in the BER pathway.
In mechanistic terms, no clear picture has yet emerged,
despite intense research conducted by many groups.
Attractive scenarios developed over the last couple of
years are (a) the localized relaxation of chromatin at the
site of DNA damage, mediated either by direct modifi-
cation of histones or noncovalent interaction of histones
with poly(ADP-ribose) present as automodification
on PARP-1 or -2; (b) a damage signalling function [3];
or (c) recruitment of specific DNA repair proteins

to the site of damage via noncovalent interaction with
poly(ADP-ribose). These are but a few of the current
hypotheses.
Recent work has added some new interesting
aspects. PARP-1 is a known interaction partner of the
Werner syndrome protein, a protein involved in DNA
repair, maintenance of genomic stability and the pre-
vention of premature ageing. Recently PARP-1 was
shown to regulate both the exonuclease and helicase
activities of the Werner syndrome protein, suggesting a
possible mechanism of action of PARP-1 [52]. Another
scenario that has been put forward is the provision of
ATP from pyrophosphorolytic cleavage of poly(ADP-
ribose). Such ATP could be used for the DNA ligation
step in BER [53]. Accordingly, the decision between
the short-patch and the long-patch pathway in BER in
living cells appears to be dependent on the availability
of ATP. The long-patch pathway would be pre-
ferred under conditions of energy shortage, as this
pathway might lead to increased generation of ATP
from poly(ADP-ribose) via increased provision of
pyrophosphate from deoxynucleotide incorporation
into DNA [54].
On the other hand, recent data clearly show that
DNA-damage induced poly(ADP-ribosyl)ation has
roles to play beyond classical BER. For instance, it
accelerates the repair of oxidative base damage as well
as of UV-induced pyrimidine dimers in a pathway
dependent on Cockayne syndrome B protein [55]. Fur-
thermore, PARP-1 plays a role in regulating double-

strand break repair, independently of p53 [56]. In this
context, a novel route for DNA double-strand breaks
rejoining seems to involve PARP-1 and XRCC1 ⁄ DNA
ligase III, i.e. proteins classically associated with
BER [57]. A particular type of DNA damage is
represented by stalled DNA topoisomerase I mole-
cules. Poly(ADP-ribose) reactivates stalled DNA topo-
isomerase I and induces resealing of the DNA strand
breaks caused by topoisomerase stalling, which may be
viewed as a direct repair activity of PARP-1 [58].
Another function of poly(ADP-ribosyl)ation in the
absence of any exogenous DNA damage was highligh-
ted recently. During spermiogenesis, spermatid differ-
entiation is marked by dramatic changes in chromatin
density and composition. The extreme condensation of
the spermatid nucleus is characterized by a shift from
histones to transition proteins and then to protamines
as the major nuclear proteins. Recently poly(ADP-
ribose) formation driven by endogenous DNA strand
breaks was discovered in spermatids of steps 11–14 of
spermiogenesis, i.e. those steps that immediately pre-
cede the most pronounced phase of chromatin con-
densation in spermiogenesis. Transient ADP-ribose
polymer formation may therefore facilitate the process-
ing of DNA strand breaks arising endogenously during
the chromatin remodelling steps of sperm cell matur-
ation [59].
It is interesting to note that there is apparently some
specificity in the downstream consequences of PARP-1
deficiency in cells surviving a genotoxic attack, in that

an increase of deletion mutations and insertions ⁄ rear-
rangements was recorded in vivo after treatment with
an alkylating agent [60]. The importance of a proficient
poly(ADP-ribosyl)ation system for the maintenance of
genomic stability and thus the prevention of cancer is
also mirrored in the finding that the V762A genetic
variant of PARP-1 [61,62], which is associated with
diminished enzyme activity, contributes to prostate
cancer susceptibility [63]. This is perfectly in line with
the diminished PARP activity previously recorded in
normal peripheral blood lymphocytes of laryngeal
cancer patients [64].
Apart from experiments aiming at inhibition of cellu-
lar poly(ADP-ribosyl)ation, experimental systems have
also been established to raise cellular poly(ADP-ribose)
levels above normal [65,66]. Supranormal levels of
cellular poly(ADP-ribose) achieved by overexpression
of PARP-1 in cultured cells proved to block genomic
instability, assessed as sister-chromatid exchange,
A. Bu
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FEBS Journal 272 (2005) 4576–4589 ª 2005 FEBS 4581
induced by DNA damage [65], thus yielding the mirror
image of what experiments with PARP inhibition have
shown. Viewed together, the data demonstrate that
poly(ADP-ribose) acts as a negative regulator of DNA-
damage induced genomic instability [67].
PARP inhibitors in cancer treatment
Proficient DNA repair is pivotal to the survival and

maintenance of genomic stability of cells and organ-
isms, given the relentless attack by endogenous and
exogenous DNA-damaging agents. In the setting of
cancer therapy with cytotoxic agents, however, DNA
repair in tumour cells will counteract the desirable cell
killing effect of the treatment. Accordingly, pharmaco-
logical PARP inhibitors, which interfere with DNA
repair pathways, have long been considered a useful
addition to current cancer chemotherapy ⁄ radiotherapy
protocols, drawing on the cocytotoxic effect of
PARP inhibition under conditions of DNA damage
[68]. Very recently, the specific killing of BRCA2-defici-
ent tumour cells by the sole administration of
PARP inhibitors, i.e. in the absence of any exogenous
DNA-damaging treatment, was demonstrated [69,70].
Apparently this phenomenon is related with the defici-
ency of BRCA2-deficient cells to perform homologous
recombination, a pathway most prominently used by
cells lacking PARP-1 activity. These findings should
allow targeting of the DNA repair defect in BRCA-
mutant cells as a new therapeutic strategy for some
forms of human cancer.
Regulation of transcription
It has long been postulated that poly(ADP-ribo-
syl)ation could influence the regulation of gene expres-
sion via regulation of chromatin remodelling [23,71].
Indeed, numerous physical and functional interactions
of PARP-1 with transcription factors have been des-
cribed [72]. PARP-1, for example, plays a pivotal role
in NF-jB-dependent gene expression, which makes it

an important cofactor in immune and inflammatory
responses [73–75]. Furthermore, recent data reveal
functions of PARP-1 in the CaM kinase IId-dependent
neurogenic gene activation pathway [76], in the
NAD
+
-dependent modulation of chromatin structure
and transcription mediated by nucleosome binding of
PARP-1 [12], and in the determination of specificity in
a retinoid signalling pathway via direct modulation of
Mediator [77]. The involvement of poly(ADP-ribo-
syl)ation in long-term potentiation in Aplysia neurones,
occurring in the absence of DNA strand breaks, has
also been linked with transcriptional effects [13].
Implications for apoptosis
The specific cleavage of PARP-1 by caspase-3 ⁄ -7
within the nuclear location signal of PARP-1 generates
a 24 kDa and an 89 kDa fragment, and this phenom-
enon has been used extensively as a biochemical mar-
ker of apoptosis. Caspase-mediated PARP-1 cleavage
is thought to cause a loss of stimulation of the cata-
lytic PARP-1 activity in the presence of DNA strand
breaks. Recently a Parp1 knock-in mouse model
(PARP-1(KI ⁄ KI)) was reported, in which the caspase
cleavage site of PARP-1 has been mutated so as to
render the protein resistant to caspases during apopto-
sis [78]. Perhaps surprisingly the mice developed nor-
mally. They also proved highly resistant to endotoxic
shock and ischaemia–reperfusion damage, which was
associated with reduced inflammatory responses in the

target tissues and cells, due to the reduced production
of specific inflammatory mediators. Despite normal
binding of NF-jB to DNA, NF-jB-mediated tran-
scription activity was impaired in these knock-in mice,
which explains the above phenotype and creates a new
and unexpected link between PARP-1 cleavage typical
of apoptosis and the regulation of NF-jB, a master
switch in inflammation.
Despite the above-mentioned PARP-1 cleavage,
massive formation of poly(ADP-ribose) can be
observed during the early stages of apoptosis indica-
ting that PARP family proteins are involved in this
process [79,80]. Furthermore it could be shown that
PARP-1 activation is required for the translocation of
apoptosis-inducing factor from the mitochondria to
the nucleus and that apoptosis-inducing factor is neces-
sary for PARP-1-dependent cell death [81].
NAD
+
depletion, necrotic cell death and
pathological conditions
Twenty years ago, a mechanism of cell death depend-
ing on the overactivation of PARP-1, and on severe
and irreversible depletion of its substrate NAD
+
, was
proposed for the first time [82]. Subsequently this para-
digm was confirmed experimentally in mammalian sys-
tems and also in plants [83]. In recent years this
mechanism has been demonstrated to play a major

role in a wide variety of pathophysiological conditions,
including ischaemia–reperfusion damage and a wide
range of inflammatory conditions (reviewed in [84,85]).
Mitochondrial dysfunction triggered by PARP-1 over-
activation seems to play a critical role for the ensuing
cell death [86]. Based on this mechanism, PARP-inhibi-
tory compounds are currently being developed as novel
therapeutics to treat such kind of diseases.
Poly(ADP-ribosyl)ation A. Bu
¨
rkle
4582 FEBS Journal 272 (2005) 4576–4589 ª 2005 FEBS
Another intriguing scenario of how the decline of
NAD
+
and the rise of nicotinamide triggered by
extensive PARP-1 activation might impact on cellular
physiology is the possible down-regulation of the activ-
ity of silent information regulator 2 (Sir2)-like mam-
malian proteins (sirtuins), a class of NAD
+
-dependent
deacetylases [87]. Sir2 activity depends on high concen-
tration of NAD
+
and is inhibited by nicotinamide.
Sirtuins have been implicated in mediating gene silen-
cing, longevity of organisms and genome stability. It
was proposed that poly(ADP-ribosyl)ation by PARP-
1, which is induced by DNA damage, could modulate

protein deacetylation by Sir2 via the NAD
+
⁄ nicotina-
mide connection [87].
Relevance of poly(ADP-ribose) catabolism
A PARG loss-of-function mutant was described in
Drosophila melanogaster, lacking the conserved cata-
lytic domain of PARG. This mutant exhibits lethality
in the larval stages at the normal developmental tem-
perature of 25 °C [88]. However, about a quarter of
the mutant fly population progressed to the adult stage
at 29 °C but then displayed progressive neurodegenera-
tion with reduced locomotor activity and a shortened
lifespan. This phenotype was accompanied by extensive
accumulation of poly(ADP-ribose) in the central ner-
vous system. These results suggest that undisturbed
poly(ADP-ribose) metabolism is required for mainten-
ance of the normal function of neuronal cells.
Recently, mouse models with mutant PARG gene
have also been created. The complete loss of PARG
activity induced by disruption of exon 1 led to the fail-
ure of cells to degrade poly(ADP-ribose) and caused
increased sensitivity to cytotoxicity and early embry-
onic lethality [89]. By contrast, combined disruption of
exons 2 and 3 led to the selective depletion of the
110 kDa isoform of the enzyme. This intervention was
compatible with survival and fertility of mice and led
to increased sensitivity to genotoxic and endotoxic
stress in vivo [90]. Viewed together a picture emerges
that loss of PARG activity may produce biological

effects that depend very much on the cellular compart-
ment(s) affected by such loss. This conclusion is rather
sobering, but not implausible in view of the multiple
cellular sites where poly(ADP-ribosyl)ation has been
detected recently (see below).
Centromere function and mitotic spindle
formation
PARP-2 together with PARP-1 has been detected
at centromeres [19,91] where they both interact with
constitutive and transient centromeric proteins [17,92],
indicating that poly(ADP-ribosyl)ation might act as a
regulator of both constitutive kinetochore proteins and
those involved in spindle checkpoint control. Whereas
PARP-2 localization is discrete at the centromere,
PARP-1 shows a broader centromeric and pericentro-
meric distribution [17]. The absence of any drastic
centromeric phenotype in Parp1 knockout mice is sug-
gestive of some functional redundancy for PARP-1 at
the centromere [19]. On the other hand, an increase in
centromeric chromatid breaks observed in Parp2
knockout mice exposed to c-irradiation has been
reported. Furthermore female-specific lethality associ-
ated with X-chromosome instability has been observed
in Parp1
+ ⁄ –
⁄ Parp2
– ⁄ –
mice [19]. These data are sug-
gestive of diverse roles of PARP-2 and PARP-1 in
modulating the structure and checkpoint functions of

the mammalian centromere, in particular during radi-
ation-induced DNA damage.
In addition, poly(ADP-ribose) was recently identified
as a new component of the spindle, in addition to the
known major spindle components including micro-
tubules, microtubule-associated proteins and motors
consisting of proteins and DNA [93]. The presence of
poly(ADP-ribose) is required for bipolar spindle
assembly and function.
Centrosomal function
The regulation of centrosome function is crucial to the
accurate transmission of chromosomes to the daughter
cells in mitosis. Both PARP-1 and PARP-3 (Table 1;
Fig. 2) have been identified at centrosomes where they
form a stable complex [20] and poly(ADP-ribosyl)ate
p53 [31]. p53, in turn, has also been shown to localize
at centrosomes and to control centrosome duplication
[94]. Thus both PARP-1 and PARP-3 seem to be
involved in centrosome duplication by modulating p53
activity via poly(ADP-ribosyl)ation. In particular,
PARP-3 localizes preferentially to the daughter centri-
ole throughout the cell cycle [20]. An attractive hypo-
thesis is that the presence of both PARP-1 and
PARP-3 at the centrosome may link the DNA damage
surveillance network to the mitotic fidelity checkpoint.
Tankyrase (Table 1, Fig. 2) is another member of
the PARP family that localizes to the centrosome in a
cell cycle-dependent manner. During mitosis, tankyrase
colocalizes with nuclear mitotic apparatus protein
(NuMa) [95], with which it was shown to form a stable

complex at the centrosome [40]. When NuMa returns
to the nucleus after mitosis this colocalization termin-
ates and tankyrase associates with GLUT4 vesicles
that coalesce around centrosomes [35] and function in
A. Bu
¨
rkle Poly(ADP-ribosyl)ation
FEBS Journal 272 (2005) 4576–4589 ª 2005 FEBS 4583
insulin-dependent glucose utilization. Thus, spindle
poles and Golgi apparatus alternately contain most of
cellular tankyrase, whereas only a small fraction of
tankyrase functions at telomeres (see below).
Structure and function of vault particles
As mentioned above, vault particles are large cytoplas-
mic RNPs. Although the cellular function of vaults is
unknown, their subcellular localization and distinct
morphology point to a role in intracellular transport,
particularly nucleo–cytoplasmic transport [96]. It was
reported that vault particles may also be involved in
intracellular detoxification, as all three vault proteins
display increased expression in many multidrug resist-
ant human cell lines examined [97]. PARP-4 is identi-
cal with the 193 kDa vault protein (Table 1, Fig. 2)
and poly(ADP-ribosyl)ates the p100 subunit (major
vault protein; MVP) within the vault particle and to a
lesser extent itself. Immunofluorescence and biochemi-
cal data show that PARP-4 is not exclusively associ-
ated with the vault particle but can also localize to the
nucleolus, the nuclear spindle and to nuclear pores
[32,96]. The enzyme has also been found in association

with mammalian telomerase but is dispensable for
telomerase function and vault structure in vivo [98].
Telomere dynamics
Parp1 knockout mice were reported to have shorter
telomeres than wild-type mice [99]. This observation is
indicative of a role of PARP-1 in maintaining telomere
length and is compatible with the positive correlation
between cellular poly(ADP-ribosyl)ation capacity (lar-
gely reflecting PARP-1 activity) and lifespan of mam-
malian species [100]. Apparently, the differences in
enzyme activity may be due, at least in part, to chan-
ges the primary structure of PARP-1 that arose during
evolution [101].
A functional role of PARP-2 in the maintenance of
telomere integrity is supported by the colocalization of
PARP-2 and telomeric-repeat binding factor 2 (TRF2),
which is a negative regulator of telomere length.
PARP-2 activity regulates the DNA-binding activity of
TRF2 via poly(ADP-ribosyl)ation of the dimerization
domain of TRF2 as well as via noncovalent binding of
poly(ADP-ribose) to the myb domain of TRF2 [102].
This protein interaction may well be involved in modu-
lating t-loop formation in response to DNA damage.
Tankyrase is another negative regulator of telomere
length [34], but surprisingly only about 10% of cellular
tankyrase protein is recruited to telomeres, and
its binding to telomeres is mediated through TRF1.
Poly(ADP-ribosyl)ation of TRF1 by tankyrase inhibits
binding of TRF1 to telomeric DNA and so contributes
to telomere length regulation, by reversing the negative

effect of TRF1 on telomere length [103]. The catalytic
activity of tankyrase is crucial for this effect, because
nuclear overexpression of tankyrase, but not of a
PARP-deficient mutant, causes the lengthening of
telomeres [104]. Additional proteins, however, are also
involved in TRF1 regulation, such as TRF1-interacting
nuclear protein 2 (TIN2), which was reported to pro-
tect TRF1 from poly(ADP-ribosyl)ation by tankyrase
via formation of a ternary complex with TRF1 and
tankyrase, yet without affecting tankyrase automodifi-
cation [105].
As mentioned above, the domain structure of tanky-
rase-2 is very similar to that of tankyrase except for
the N-terminal HPS domain, which is missing in
tankyrase-2 (Fig. 2) [39,41]. Likewise the two enzymes
share several interaction partners including IRAP,
TAB182 and TRF1. Overexpression of either tanky-
rase or tankyrase-2 in the nucleus released endogenous
TRF1 from the telomere, suggesting that the function
of the two enzymes may partially be redundant
[103,104].
Taken together, at least four members of the PARP
family have been implicated in telomere regulation. A
full understanding of their distinct functions at telo-
meres, their regulation and possible functional cooper-
ation will require a substantial amount of additional
research work.
Trafficking of endosomal vesicles
Despite the effect of tankyrase on telomere dynamics,
it should be noted that most of this protein is found

outside the cell nucleus. As mentioned above, it can
either be detected at centrosomes, where it seems to
interact with NuMa [40,95], or in association with
nuclear pore complexes [103] or at Golgi-associated
GLUT4 vesicles [35]. Tankyrase was also reported to
interact with a tankyrase-binding protein of 182 kDa
(TAB182), displaying a heterochromatin-like staining
pattern in the nucleus and colocalizing with cortical
actin in the cytoplasm [106]. Endocytotic vesicles in
myocytes and adipocytes contain the glucose transpor-
ter GLUT4 as well as IRAP. The reversible transloca-
tion of GLUT4 between these GLUT4 vesicles in the
Golgi and the plasma membrane allows insulin to
regulate glucose utilization. Tankyrase appears to be
an important insulin-signalling target, as the protein
not only interacts with IRAP located to GLUT4 stor-
age vesicles in the Golgi, but is also phosphorylated by
mitogen-activated protein kinase (MAPK) upon insulin
Poly(ADP-ribosyl)ation A. Bu
¨
rkle
4584 FEBS Journal 272 (2005) 4576–4589 ª 2005 FEBS
stimulation. Tankyrase poly(ADP-ribosyl)ates IRAP,
as well as itself, and this activity is enhanced
by MAPK-mediated phosphorylation indicating that
tankyrase may mediate the long-term regulation of
GLUT4 vesicles by the MAPK cascade [35,107].
Emerging new PARP family members
TIPARP (PARP7)
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a pro-

totype substance from the class of dioxins and causes
pleiotropic effects in mammalian species through
modulating gene expression. A novel, TCDD-inducible
member of the PARP family (TiPARP or PARP7 [2])
was recently identified and characterized (Table 1)
[108,109]. TiPARP mRNA is expressed in a broad
range of mouse tissues and can poly(ADP-ribosyl)ate
histones. Genetic analyses revealed that induction
depends on the aromatic hydrocarbon receptor (AhR)
and on the aromatic hydrocarbon receptor nuclear
translocator (Arnt).
PARP10
Recently a novel member of the PARP family (PARP-
10) has been characterized at the biochemical
level, which is a novel c-myc-interacting protein with
poly(ADP-ribose) polymerase activity (Table 1) [110].
PARP-10 is a 150 kDa protein that interacts with Myc
and possesses a domain with homology to RNA recog-
nition motifs. PARP-10 can poly(ADP-ribosyl)ate itself
and core histones, but neither Myc nor Max, a well-
known c-myc interactor. PARP-10 is localized to the
nuclear and cytoplasmic compartments under the con-
trol of a nuclear export sequence, which also seems to
be relevant for the inhibitory effect of PARP-10 con-
cerning c-Myc- and E1A-mediated cotransformation of
rat embryo fibroblasts.
Conclusion and outlook
The field of poly(ADP-ribosyl)ation is currently a very
exciting one and it has widened in every respect.
Whereas until a couple of years ago a single enzyme

was looked at (PARP-1), we are now dealing with over
a dozen. Whereas DNA strand breakage used to be
considered the only trigger of poly(ADP-ribose) syn-
thesis and consequently the only relevant cellular
condition for poly(ADP-ribose) function, several
additional activation conditions and cellular pheno-
mena related with poly(ADP-ribosyl)ation have been
described. As a consequence, the range of specialists
interested in poly(ADP-ribosyl)ation has broadened
enormously. Obtaining a comprehensive picture of the
biological functions of poly(ADP-ribosyl)ation and the
underlying molecular mechanisms is highly desirable
and would further accelerate the transfer of basic sci-
entific information on this subject to the medical appli-
cation. Hopes that this may be achieved in the not too
distant future are increasing in view of the growing
interest of the scientific community in this field.
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