Poly(ADP-ribose) : The most elaborate metabolite of NAD+

MINIREVIEW

Poly(ADP-ribose)

The most elaborate metabolite of NAD+

Alexander Burkle

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. Burkle, Department of Biology,

Box X911, University of Konstanz,

D-78457 Konstanz, Germany

Tel: +49 7531 884035

Fax: +49 7531 884033

E-mail: [email protected]

Website: http://gutenberg.biologie.

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+

PAR

PAR

n NAD+

n Nicotinamide + n H+

Rib

O

O

PO

O

PO

Rib O-

O-

N

N

NH2

N

NAcceptor protein CH2 C

O

O-

Rib

N

N

NH2

N

N

O

O

O-

PO

O

O-

PO

Rib

Rib

N

N

NH2

N

N

O

O

O-

PO

O

O-

PO

Rib

RibRib

N

N

NH2

N

N

O

O

O-

PO

O

O-

PO

Rib

Rib

N

N

NH2

N

N

O

O

O-

PO

O

O-

PORib Rib

N

N

NH2

N

N

O

O

O-

PO

O

O-

PO

Rib Rib

N+

H2NCO

N

H2NCO

+ H+

Rib

N

N

NH2

N

N

O

O

O-

PO

O

O-

PO

Rib

+

CH2 C

O

O

CH2 C

O

OAcceptor 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. Burkle Poly(ADP-ribosyl)ation

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)


[PARP-2, NAD+ poly(ADP-ribose) polymerase-2]

14q11.2-q12 10038 583 aa (66 kDa)


[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, 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 BRCTNLSZFI ZFII

Module A B C E D F

DNA-binding domain (DBD) NAD+-binding domain

Automodification domain

Active site

DBD

NLS

BRCT

SA24 ankyrinHPS

SA24 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. Burkle


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-PKcs). 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



interfered with the G1 ⁄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.



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,



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 ⁄ radiotherapyprotocols, 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-dependentneurogenic 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 ⁄ -7within 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.



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



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



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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Poly(ADP-ribose) : The most elaborate metabolite of NAD+