DNA Repair 3 (2004) 11031108

Review

PARP-1, PARP-2 and ATM in the DNA damage response: functional synergy in mouse development
Aline Huber, Peter Bai, Josiane Mnissier de Murcia, Gilbert de Murcia
Unit 9003 du CNRS, Ecole Suprieure de Biotechnologie de Strasbourg, Boulevard Sbastien Brant, BP 10413, 67412 Illkirch Cedex, France Available online 23 June 2004

Abstract Poly(ADP-ribosyl)ation is an immediate DNA damage-dependent posttranslational modication of histones and nuclear proteins that contributes to the survival of injured proliferating cells. Poly(ADP-ribose) polymerases (PARPs) now constitute a superfamily of 18 proteins, encoded by different genes and displaying a common conserved catalytic domain. PARP-1 (113 kDa), the founding member, and PARP-2 (62 kDa) are both involved in DNA-break sensing and signaling when single strand break repair (SSBR) or base excision repair (BER) pathways are engaged. The generation by homologous recombination of decient mouse models have conrmed the caretaker function of PARP-1 and PARP-2 in mammalian cells under genotoxic stress. This review summarizes our present knowledge on their physiological role in the cellular response to DNA damage and on the genetic interactions between PARP-1, PARP-2, Atm that play an essential role during early embryogenesis. 2004 Elsevier B.V. All rights reserved.
Keywords: PARP-1; PARP-2; ATM

1. PARP-1 and PARP-2, two DNA damage-dependent enzymes of a large superfamily The presence of DNA strand breaks in the cells of higher eukaryotes activate signal transduction pathways that trigger cell cycle arrest and repair mechanisms leading ultimately to cell survival or programmed cell death. Central to pathways that maintain genomic integrity is the immediate modication of histones and nuclear proteins by ADP-ribose polymers catalyzed by poly(ADP-ribose) polymerases (PARPs) [1]. A large repertoire of 18 sequences encoding novel PARPs now extend considerably the eld of poly(ADP-ribosyl)ation reactions to various aspects of the cell biology including cell proliferation, cell death and energy metabolism. The members of the PARP superfamily are characterized by a conserved core responsible for the catalytic activity to which a number of specic targeting and regulatory modules have been added. This modular architecture of PARPs lead to a plethora of possible functions, perhaps broader than genome surveillance [2]. PARP-1 and PARP-2, are so far the sole members whose catalytic activity stimulated in vitro and in vivo by DNA
Corresponding author. Tel.: +33 388 90 24 47 07; fax: +33 388 90 24 46 86. E-mail address: demurcia@esbs.u-strabg.fr (G.de Murcia). 1568-7864/$ see front matter 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2004.06.002

strand-breaks [3,4] catalyze the transfer of the ADP-ribose moiety from the respiratory co-enzyme NAD+ to a limited number of acceptor proteins involved in chromatin architecture and in DNA metabolism (Fig. 1A). PARP-1 (113 kDa) is a multifunctional enzyme, highly conserved from human to A. thaliana, but absent in yeast. Its domain structure comprises (i) a N-terminal DNA break recognition module containing a duplicated zinc nger, homologous to that of DNA ligase III (ii) a central BRCT motif, present in a large number of proteins involved in the maintenance of genomic integrity and cell cycle checkpoints, acting as the major proteinprotein interacting interface and (iii) a carboxy-terminal region bearing all the different catalytic activities associated with the full-length enzyme: NAD+ hydrolysis and initiation, elongation, branching and termination of ADP-ribose polymers (Fig. 1B). The basal activity of PARP-1 is stimulated 500 times by the presence of breaks in the DNA. The catalytic domain is by far the most evolutionarily conserved region. It contains a block of 50 amino acids (aa 859908), the PARP signature, virtually unchanged from human to plants [1]. It is conserved to various degree among the 18 human PARP orthologs [2]. PARP-2 (62 kDa) was discovered as a result of the presence of residual DNA-dependent PARP activity in embryonic broblasts derived from PARP-1-decient mice [3]. PARP-2 is responsible for only 1015% of the total PARP

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Fig. 1. (A) Metabolism of poly(ADP-ribose) during DNA damage and repair induced by various genotoxins. (B) The domain structure of the two DNA damage-dependent human poly(ADP-ribose) polymerases-1 and -2 (PARP-1, PARP-2). PARG, poly(ADP-ribose) glycohydrolase; NLS, nuclear localization signal. BRCT; BRCA1 C-terminus motif. Nam; nicotinamide.

activity fully stimulated by DNA strand-breaks in a cellular extract. Its catalytic domain has the strongest resemblance among all the other family members to that of PARP-1 with 69% similarity. Structural studies of the murine PARP-2 demonstrated that its catalytic domain is very similar to that of PARP-1 except in the vicinity of the acceptor site that is most probably specic for protein substrates [5]. PARP-2 is a nuclear protein that binds to and is activated in vitro by DNase-I treated DNA. However, its DNA binding domain differs from that of PARP-1 and targets DNA gaps but not nicks (Am et al., in preparation). The N-terminal domain of PARP-2, 75 aa in size, is responsible both for the localization of PARP-2 to the nucleus (Fig. 1B) and the recognition of DNA interruptions. It displays homology with the SAP domain found in various nuclear proteins like APE-1 and Ku70, involved in chromosomal organization and in DNA repair. Interestingly, this domain is also found in plant PARP-2, whose transcription is dramatically upregulated in response to a sublethal dose of ionizing radiation (IR) [6]. PARP-2 acts as a chromatin modier too but poly(ADPribosyl)ates histone H2B while PARP-1 targets histone H1. PARP-2 interacts with PARP-1, they share common

partners involved in the single strand break repair (SSBR) and base excision repair (BER) pathways: XRCC1, DNA polymerase- , and DNA ligase III [4] suggesting that they are both engaged in the same DNA repair complex. PARP-1 and PARP-2 interact also with proteins involved in the kinetochore structure and in the mitotic spindle checkpoint. However, specic partners of PARP-2 are begining to be discovered such as the telomeric protein TRF2 suggesting a link with the control of telomere integrity [7].

2. Poly(ADP-ribosyl)ation, the rst line of defense in response to DNA breaks: for better or for worse Several characteristics of PARP-1 behavior upon DNA damage classify this fascinating enzyme as a component of the early response to DNA strand breaks. Recent studies from Okano et al. [8] and El-Khamisy et al. [9] as well as work from our laboratory [10] reveals that the immediate synthesis of PAR at a DNA strand break constitutes an initiating event in a damaged cell. This, triggers the subsequent co-ordination of DNA strand-breaks detection by PARP-1

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Fig. 2. Scheme depicting the involvment of PARPs in the repair of single strand breaks process. (i) Following SSB dection by PARP-1, poly(ADP-ribose) synthesis at the DNA damage site triggers the immediate recruitment of XRCC1 and the assembly of the SSBR repair complex [37]. (ii) Polynucleotide kinase (PNK), stimulated by XRCC1, converts the DNA ends to 5 -phosphate and 3 -hydroxyl moieties that (iii) enables DNA Pol , stimulated by PARP-1, to ll the gap. The size of the repair patch is controlled by the nuclease FEN1 and PARP-2. (iv) The ligation step is nally catalyzed by DNA Lig III whose stability is controlled by XRCC1. Closing of the break then abrogates PARPs activities.

and signaling to the SSBR pathway, the whole process being performed in less than 15 s. PARP-1 is particularly well suited to fulll several key functions following the occurrence of an interruption of the sugar phosphate backbone: (i) efcient sensing of the DNA break [11,12]; (ii) translation and amplication of the damage signal in a posttranslational modication of PARP-1 itself (automodication) and of histones H1 and H2B (heteromodication) that triggers chromatin structure relaxation [13] and increases the access to the break; (iii) PAR-dependent recruitment of XRCC1 to the damaged site [8] mediated by its BRCT1 motif that displays high afnity to PAR and PARP-1 [14,15]. Although not essential in vitro, XRCC1 as a scaffold protein with no known enzymatic function plays a critical role in the co-ordinated handling of the damaged DNA, from one repair enzyme to the next, in the BER pathway [16] (see Fig. 2). It is nevertheless essential during mouse development [17]. The absence of PARP-1 or the inhibition of its enzymatic activity totally prevents the dynamic recruitment of XRCC1 to the break, thus explaining the important delay in strand-break rejoining that causes

a severe DNA repair defect in PARP-1/ damaged cells or in damaged cells treated with 3-AB. However, the repair defect observed in PARP-2/ damaged cells [4] cannot be attributed to a lack of XRCC1 recruitment, since this step still occurred following DNA breakage [10]. Rather, PARP-2 activity seems to be associated to a subsequent step in the SSBR pathway (Fig. 2). PARP-1 activity induced by high levels of DNA breaks renders PARP-1 a risky cellular factor when the genome is being degradated during cell death. Therefore, to limit futile DNA repair during apoptosis and to preserve the NAD+ and ATP pools, PARP-1 is inactivated by a caspase-dependent cleavage [18]. A quite different scenario takes place in acute pathophysiological conditions such as necrosis or caspase-independent cell death (chromatinolysis) where PARP-1 is instrumentalized by the Apoptosis-Inducing factor (AIF) a avoprotein normally conned to the mitochondrial intermembrane space. In response to DNA injury, AIF in concert with the EndoG nuclease translocates rapidly to the nucleus and degradates the chromatin into 50 kb fragments [19]. This large scale DNA fragmentation overactivates PARP-1 and kills the cells in a caspase-independent manner, thus leading to inammatory injury in the corresponding tissue [20,21]. Dawson and colleagues provided evidence that PARP-1 initiates a nuclear signal that propagates to mitochondria and triggers the release of AIF that leads to cell death. Accordingly, PARP-1-decient cells or pharmacologically inhibited wild-type cells are fully protected against excessive damage. Clearly, the activation of PARP-1 has two opposed meanings depending upon the cell type and the extend of the DNA damage: (i) Survival: in replicating cells, limited damage to DNA induces PARP activity and stimulates 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, but the molecular determinants that switch between DNA repair and cell-cycle arrest or apoptosis are not yet fully understood. When apoptosis is induced, PARP-1 and -2 as survival factors are cleaved by caspases and are thus inactivated. (ii) Cell death: In post-mitotic cells, reactive oxygen species (ROS) damage DNA activate PARP and trigger AIF translocation to the nucleus. The resulting DNA fragmentation overactivates PARP and leads to nuclear condensation. Understanding the switch between these two facets of PARP biology might ultimately improve pharmacological strategies to enhance both antitumor efcacy as well as the treatment of a number of inammatory and neurodegenerative disorders.

3. PARP-1- and PARP-2-decient mice are highly sensitive to DNA damage To gain further insight into the functional importance of DNA-dependent poly(ADP-ribosyl)ation reactions at the

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animal level, we and others have generated PARP-1/ [2224] and PARP-2/ decient mice [25]. Both mutant mice are similarly radiosensitive and display severe genomic instability after -irradiation. Mouse embryonic broblasts (MEFs) lacking either PARP-1 [24,2628] or PARP-2 [4] display a similar severe defect in alkylation induced DNA strand-break resealing conrming that PARP-1 and PARP-2 are involved in the maintenance of genomic integrity. Interestingly, the sensitization of PARP-2/ MEFs is accompanied by a G2/M accumulation, the acquisition of 8N DNA content, the presence of anaphase bridges, and the occurrence of preferential breaks in centromeric and subcentromeric regions reecting kinetochore defects. Thus, although PARP-2-specic activity is reduced compared to that of PARP-1, the phenotype of the PARP-2/ and PARP-1/ mice are equally severe, because they both participate not in the catalytic steps of the process, but instead they dramatically affect the efciency and/or the accuracy of the repair. The double null mutant PARP-1/ ; PARP-2/ embryos are not viable and die around E7.5 at the onset of gastrulation, demonstrating that the expression of both PARP-1 and PARP-2 and/or DNA-dependent poly(ADP-ribosyl)ation is essential during early embryogenesis. Interestingly, a specic female embryonic lethality is observed in PARP-1+/ PARP-2/ mutant at mid-gestation (E9.5). Metaphase analyses of E8.5 embryonic broblasts highlight a specic instability of the X-chromosome in those females but not in males. [25]. These results support the notion that, even if a critical level of the DNA repair capacity is reached in PARP-1+/ PARP-2/ mutant, avoiding an early lethality, the poor DNA repair efciency may lead to a severe genomic instability, detrimental rst to the females.
Fig. 3. External views and histological sections of E8.0 normal (a, c) (N, normal and PARP-1/ ; Atm+/ ), (b, d) (PARP-1; Atm) double null concepti (/; /), (e, f) (PARP-2; Atm) double null concepti (/; /). Abbreviations: A, amnion; AL, allantois; EC, ectoplacental cavity; EX, extraembryonic material; EM, embryo; F, foregut pocket; H, head folds; P, placenta; S, somites; YC, yolk sac cavity. (Taken from ref. [38] with permission.)

4. PARP-1, -2, Atm: genetic interactions in mouse development The multifunctional protein kinase Atm is an orchestrating factor in the cascade of events leading to activation of the cellular response to DNA double strand breaks, which includes repair and cell-cycle checkpoint pathways [29] (see also Kurz and lees-Miller, in press). ATM is missing or inactivated in patients with the genomic instability syndroma ataxia-telangiectasia (A-T) (Chun and Gatti, in press). Cell lines established from Atm-decient mice, like those from A-T patients exhibit genomic instability and defective response to double strand breaks (DSBs), e.g., defective activation of the cell cycle checkpoints after ionizing radiation [30] likely attributed to the defect in sensing, repairing and signaling DSBs. Beside a clear defect in DSB response, A-T cells may chronically suffer from increased oxidative stress [31]. The relationship between ATM and PARP-1 on one hand and ATM and PARP-2 on the other hand were so far unexplored in the mice. Two mouse models decient for ATM

and PARP-1 (Atm/ ; PARP-1/ ) or PARP-2 (Atm/ ; PARP-2/ ) were generated to investigate the impact of these putative interactions on genomic stability and embryonic development. No homozygous PARP-1, Atm and PARP-2, Atm-disrupted pups were observed (Fig. 3 and Table 1) implying that a potent synergistic interaction exists between Atm and PARP-1 as well as Atm and PARP-2 that impacts on the embryonic development. The double-null mutants embryos die early in development, subsequent to the gastrulation stage. During this restrictive developmental window, undifferentiated stem cells that form the epiblast sustain high cell division rate. In proliferative state, intense metabolism during embryonic growth is accompanied by an oxidative burst that

A. Huber et al. / DNA Repair 3 (2004) 11031108 Table 1 Genotype analysis of (PARP; Atm) embryos from heterozygous matings Intercross Age of progeny n No. of embryo (% of total) with Atm genotype +/+ PARP-1/ ; Atm+/ Newborn E8.0E11.5 Newborn E8.5 123 60 163 38 30 14 59 10 (24) (24) (36) (26) +/ 93 29 104 19 (76) (48) (64) (50)

1107

/ 0 17 (28)a 0 9 (24)a

PARP-2/ ; Atm+/
a

Growth retarded embryos.

may cause oxidative damage to genomic DNA. This feature would be exacerbated in the double mutant embryos by the perturbation of the cellular balance of reactive oxygen species which leads to constant oxidative stress in Atm-decient cells [31]. Alternatively, the lower rate of repair that we and others have previously shown in PARP-1/ [26,27] and in PARP-2/ [4] cells combined to Atm deciency may have seriously weakened the cells defense against endogenous DNA damage, increasing the likelihood of lethal chromosomal breaks and replication failure at a stage of development accompanied by rapid cell division.

5. Concluding remarks The phenotype displayed by each combinations of double mutants are more severe than either of the single knockout (Fig. 4) suggesting that Atm, PARP-1 and PARP-2 participate in overlapping DNA damage signaling pathways or regulate distinct forms of DNA repair that partially compensate for each other. Interestingly, breeding experiments aimed at generating decient mice in both Ku80 and PARP-1 [32] and Ku80 and Atm [33] also lead to early embryonic lethality at the gastrulation stage. Thus, the loss of certain components of the SSBR/BER and DSB repair pathways lead to massive apoptosis of the embryo at the onset of gastrulation perhaps as a result of unrepaired DNA damage or due to a defect in DNA-damage signaling. Interestingly, both PARPs, Atm and DNA PKcs are DNA damage-dependent chromatin modiers. In response to DNA breaks, PARP-1 and PARP-2 poly(ADP-ribosyl)ate histone H1 and H2B, causing the immediate relaxation of the 30 nm chromatin ber, a necessary step to increase the access to the break [13,34]. Upon -irradiation, the activation of the Atm kinase by autophosphorylation

induced-monomerization has been shown recently to be sensitive to conformational changes of chromatin [35] leading to histone H2AX phosphorylation of its C-terminal tail on Ser-139. H2AX phosphorylation is now believed to participate in chromatin reconguration and foci formation that serve to increase the local concentration of DSB repair factors and/or limit the diffusion of the broken DNA ends until the break is repaired [36] (Fig. 5). Given that changes in chromatin structure emanating from DNA breaks are probably the most initiating events in the cellular response to DNA damage, it is tempting to speculate that histone modications play an essential role, not only in DNA packaging, but also monitoring the integrity of the mammalian genome. The disruption of both PARPs and Atm routes to chromatin may also explain the impossibility for the resulting embryo to develope normally.

Fig. 4. Early embryonic lethality and genetic interactions between PARP-1, PARP-2, Atm and Ku80 (see text for references).

Fig. 5. Schematic representation of the DNA strand-breaks signaling and processing pathway. The pathway is triggered by IR resulting in single- and double-strand breaks that immediately initiate two posttranslational modications of histones and nuclear proteins catalyzed by DNA damage-dependent PARPs and Atm. Poly(ADP-ribosyl)ation of histones H1 and H2B increase chromatin accessibility at the actual site of DNA damage thus promoting DNA repair. Phosphorylation of histone H2AX by Atm occurs at or near the double-strand break and is required for the phosphorylation of 53BP1 that participates to nuclear foci organization and the subsequent recruitment of several Atm downstream targets.

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Acknowledgements We thank A. Wynshaw-Boris for the Atm-decient mice. P.B. was supported by a FEBS long-term fellowship. This work was supported by funds from Centre National de la Recherche Scientique, Association pour la Recherche Contre le Cancer, Electricit de France, Ligue Nationale Contre le Cancer and Commissariat lEnergie Atomique.

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