Ionizing radiations in induce poly(ADP-ribosyl)ation, a conserved DNA-damage response essential for...

DNA Repair 4 (2005) 814–825

Ionizing radiations inCaenorhabditis elegansinducepoly(ADP-ribosyl)ation, a conserved DNA-damage

response essential for survival

Florence Dequen, Steve N. Gagnon, Serge Desnoyers∗

Pediatrics Research Unit, CHUQ-CHUL Research Centre, Department of Pediatrics, Laval University, Sainte-Foy, Que., Canada

Received 7 July 2004; received in revised form 19 April 2005; accepted 26 April 2005Available online 31 May 2005

Abstract

Poly(ADP-ribosyl)ation is one of the first responses to DNA damage in mammals. Although it is involved in base excision repair, itsexact role has not been ascertained yet. Poly(ADP-ribose) polymerase-1 (PARP-1) and PARP-2 mediate most of the poly(ADP-ribosyl)ationresponse in mammals and are well conserved in evolution. Their respective homologues PME-1 and PME-2 are found in the nematode

port theplayed aortance, DHQ,

hibitor 3ABas very

lar patternolymerst and very

rkse.

Caenorhabditis elegans, a well-known genetically tractable model currently used in DNA damage response research. Here we refunctional analysis of PME-1 and PME-2 in presence of DNA damage. Worms irradiated with high doses of ionizing radiations dissharp drop in their NAD+ content immediately after treatment, and a biphasic increase in poly(ADP-ribose). The physiological impof the poly(ADP-ribosyl)ation response was highlighted when worms were preincubated with mammalian PARP inhibitors (3ABPJ34) and irradiated. The embryonic survival rate of the progeny was significantly decreased in a dose-dependent manner. The inhad a weak effect on embryonic survival, followed closely by DHQ. However, PJ34, a member of the phenantridinone family, weffective even when used at low concentration (100 nM). In vitro PARP assay using recombinant PME-1 and PME-2 showed a simiof inhibition where 3AB and DHQ were weak inhibitors, and PJ34 a stronger one. Inhibitors affect mostly the poly(ADP-ribose) pelongation at high concentrations. These results suggest that poly(ADP-ribosyl)ation in response to DNA damage is an ancienimportant biochemical process protecting DNA from deleterious modification.© 2005 Elsevier B.V. All rights reserved.

Keywords:Post-translational modification; Nematode; Ionizing radiation; PARP; PARP inhibitors; Survival rate

Abbreviations: 3AB, 3-aminobenzamide; ATM, ataxia telangectasiamutated; DBD, DNA binding domain; DHBB, dihydroxyboronyl Bio-Rex; DHQ, 1,5-dihydroxyisoquinoline; DMSO, dimethyl sulfoxide;

1. Introduction

Cells have elaborated complex molecular netwothrough evolution to maintain the integrity of their genom

DNA-PK, DNA-dependent protein kinase; EDTA, ethylene diaminetetraacetic acid; IPTG, isopropylthiogalactoside; IR, ionizing radiation;pADPr, poly(ADP-ribose) polymer; PARP, poly(ADP-ribose) polymerase;PARG, poly(ADP-ribose) glycohydrolase; PBS-TM, phosphate buffered

In mammals, one of the first cellular response to DNA strandbreaks is a dramatic increase in poly(ADP-ribosyl)ation[1]. This posttranslational modification is largely mediated

on-in

ain

meD)

saline-Tween milk; PJ34,N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide HCl; PME, poly(ADP-ribose) metabolism enzyme;PMSF, phenylmethylsulphonylfluoride; NAD, nicotinamide adenine din-ucleotide; TEN, Tris/EDTA/NaCl; TiPARP, 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible PARP; VPARP, vault PARP

∗ Corresponding author. Present address: Pediatrics Research Unit, RoomT2-48, CHUL Research Centre, 2705 Laurier Blvd, Sainte-Foy, Que.,Canada G1V 4G2. Tel.: +1 418 654 2296; fax: +1 418 654 2761.

E-mail address:[email protected] (S. Desnoyers).

by poly(ADP-ribose) polymerase-1 (PARP-1), the canical member of the PARP family. This family of proteincludes among others PARP-2[2], PARP-3 [3], VPARP[4], tankyrase-1 and -2[5,6], and Ti-PARP[7] which arestructurally divergent members harboring a catalytic domknown as the PARP signature[8].

The 113-kDa enzyme PARP-1 is a modular enzycomposed of a N-terminal DNA binding domain (DB

1568-7864/$ – see front matter © 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.dnarep.2005.04.015

F. Dequen et al. / DNA Repair 4 (2005) 814–825 815

including two zinc-finger motifs, a central automodificationdomain and a C-terminal catalytic domain[8]. PARP-2 iscomposed of a basic DNA binding domain, without zincfingers, and a catalytic domain[2]. PARP-1 uses NAD+

molecules to catalyze the covalent attachment of ADP-riboseunits to an assortment of target proteins including itself andhistones [9]. Poly(ADP-ribosyl)ation dramatically altersthe functions of target proteins, and regulates many nuclearfunctions such as, DNA replication, genomic stability, celldeath, transcription, and DNA repair[9–13]. For example,when chromatin proteins are modified with poly(ADP-ribose), disruption of their macromolecular complexes anddecondensation of chromatin will occur. In turn, this willallow repair enzymes to perform their task, especially duringthe base excision repair process[14].

PARP-1 activity is kept to a minimum in normal condi-tions of growth. Therefore, the polymers size is short, andrange from single residues to oligo(ADP-ribose) units. How-ever, once PARP-1 makes contact with strand breaks via itszinc finger, its enzymatic activity is stimulated by a factorof up to 500[8,15]. This leads to the formation of long, lin-ear or branched polymers of ADP-ribose (poly(ADP-ribose))reaching 200 units in size[16]. Therefore, PARP-1 is consid-ered to be a DNA nick sensor[15]. DNA damage recognitionand signaling are also mediated by cell cycle checkpoint con-trol proteins such as p53, DNA protein kinase (DNA-PK),aa t rolef ons[ e-p esh anti exactr .

int dm lec-u rr lisme -1 RP-1 eDb func-t ger r-t n ag

2

2

nds ndardt

2.2. Irradiation

Exposure to gamma rays was performed in a137Cs Gam-macell extractor (Nordion International, Inc.) at a dose rate of∼1 Gy/min at room temperature. For biochemical determina-tions, worm pellets (5–6 ml) were irradiated at 60 and 120 Gy.Worm pellets were then flash-frozen in liquid nitrogen 0, 5,15, and 30 min after irradiation, stored at−80◦C, and used forquantification of NAD+, poly(ADP-ribose) and for Westernblot. For embryonic survival determination, worms treatedwith PARP inhibitors and grown in 96-well plates received adose of 120 Gy.

2.3. Processing of worm pellets

Worm pellets were grinded into a fine powder in liquidnitrogen using a mortar and a pestle. The powder was trans-ferred into a cold 50 ml tube, mixed and homogenized in15 ml ice-cold 1N perchloric acid for 1 min and precipitatedon ice for 45 min. It was then centrifuged at 2500 rpm for15 min at 4◦C[26]. The supernatant (S1) was used for NAD+

determination and the pellet (P1) for DNA determination andpoly(ADP-ribose) isolation.

Supernatant (S1) was further processed in preparationfor NAD determination. Five millilitres of S1 was neutral-ip cip-i ach1 mina[

nol( d byca wast -c red a-t n fort B)r

2

pu-r BBr -rc r at3 duesw -tb H7 fore

taxia telangectasia mutated protein (ATM)[17,18]. PARP-1cts upstream of p53, and was shown to play an importan

or cell survival in early step response to ionizing radiati19,20]. PARP-2 poly(ADP-ribosyl)ation activity is also dendent on DNA strand breaks[21]. Therefore, both enzymave a similar function in the cell and are partially redund

n the DNA damage response process. However, theirole in DNA damage response and repair is still debated

The evolutionary study of DNA damage responsehe nematodeCaenorhabditis eleganshas recently provideany answers regarding the minimal composition of molar pathways leading to cellular response[22]. We earlieeported the presence of the poly(ADP-ribose) metabonzyme (pme) in the worm[23,24]. We also identified PMEand PME-2 as structural orthologues of mammalian PAand PARP-2, respectively[23]. However, their role in thNA damage response pathway inC. elegansremains toe determined. In the present study, we undertook the

ional analysis of PME-1 and PME-2 during DNA damaesponse inC. elegans. We report the physiological impoance of poly(ADP-ribose) metabolism for the worm ienotoxic context.

. Materials and methods

.1. C. elegans strains and culture

C. elegansN2 (Bristol strain) were handled, cultured, ataged as previously described and according to staechniques[25].

zed with 2.5 ml of 2N KOH, 0.33 M KPO4, pH 7.5. TheH was adjusted at 7.5 with 1N PCA if required. Pre

tate was obtained after leaving 30 min on ice (vortex e5 min) and removed by centrifugation at 2500 rpm for 5t 4◦C. The supernatant (S2) was stored at –80◦C until use

26].The pellet (P1) was washed twice with 20 ml etha

100%) and twice with 20 ml petroleum ether, recovereentrifugation at 2500 rpm for 10 min at 4◦C, dried for 2 ht room temperature and grinded into a fine powder. It

hen mixed with 10 ml of 1 M KOH–50 mM EDTA and inubated 90 min at 60◦C. At this point, all solid matters weissolved and a 500-�l aliquot was taken for DNA determin

ion (see below). The pH was adjusted to 9.0 in preparatiohe chromatography on Dihydroxyboronyl Bio-Rex (DHBesin.

.4. Dihydroxyboronyl Bio-Rex affinity chromatography

Samples containing poly(ADP-ribose) polymers wereified using DHBB chromatography. Preparation of DHesin was done as described previously[26,27]. Chromatogaphy was performed as described by Shah et al.[26] ex-ept for the elution, which was done with 4 ml of wate7◦C. The eluates were desiccated O/N. The dry resiere dissolved in 20�l of water for pADPr determina

ion by immunodot blot, or dissolved in 40�l of loadinguffer (50% (w/v) urea, 25 mM NaCl, 4 mM EDTA (p.5), 0.02% xylene cyanol, 0.02% bromphenol blue)lectrophoresis.

816 F. Dequen et al. / DNA Repair 4 (2005) 814–825

2.5. Quantification of poly(ADP-ribose) by immunodotblot and analysis

pADPr samples (1�l) were diluted in 0.4 M NaOH,10 mM EDTA and loaded on an hybond N+ membrane aspreviously described[28]. The membrane was saturated 1 hin PBS-TM (PBS, pH 7.4; 5% non fat dried milk; 0.1% tween20) and incubated with anti-pADPr antibody (LP9610)[28]diluted 1:10,000 in PBS-TM overnight at room temperature.The membrane was washed with PBS-TM and incubatedwith peroxidase-conjugated anti-rabbit IgG second antibodydiluted 1:2500 in PBS-TM, 30 min at room temperature.Detections was revealed by cheluminescence (Perkin-Elmerwestern lightning kit). The integrated optical density ofthe samples were compared to a standard curve made outof purified pADPr [28]. Samples from worm pADPr andbovine pADPr were digested with 5�U of bovine PARG indigestion buffer (50 mM KPO4, pH 7.5, 50 mM KCl, 10 mM�-mercaptoethanol, 0.1 mM PMSF) overnight at 37◦C priorto immunodetection[29].

2.6. Quantification of NAD+ by microassay

The assay was made in a 96-microwell flat bottom as-say plate[26]. Samples (25 or 50�l) were combined withreagent mix (final concentrations: 0.1 M bicine pH 7.8, 0.5 Me ma 5-d inee al-c 0f1 tionsod

2

t dyem Eu-g MN hed platebp and4 de-t m 0t

2

ms( ormsr n aFR de-

termined by the method of Bradford[32]. Equal amounts(400�g) of protein were loaded on SDS-PAGE and trans-ferred to a nitrocellulose membrane. Membrane was blockedwith PBS-TM, then incubated overnight at room tempera-ture with anti-pADPr antibodies (LP96-10) diluted 1:10,000in fresh PBS-TM. After three washes with PBS-TM, mem-brane was incubated with anti-rabbit IgG conjugated withperoxidase, diluted 1:2500 in PBS-TM, for 30 min at roomtemperature. Detection was done by chemiluminescence onX-ray film. The resulting films were further analyzed using aquantitative densitometric analysis. Each lane were attributedan integrated optical density number. This analysis was doneusing a BioImage Visage 110s workstation and with the NIHimage software.

For anti-His detection in bacterial extracts, an equalamount of extracts (20�g) was loaded on two separate 10%polyacrylamide gels, one for Coomassie staining and theother for western blot analysis. Once transferred to nitro-cellulose membrane and blocked with PBS-TM, extractswere probed with anti-polyhistidine mouse antibody (SigmaChemical Co., St. Louis, MO) diluted 1:2000 in PBS-TM. Af-ter three washes with PBSTM, membrane was incubated withanti-mouse IgG conjugated with peroxidase, diluted 1:5000in PBSTM, for 25 min at room temperature. Detection wasdone by chemiluminescence

2

late(cP erea ks O,a rmsw tem-p

2

de-s ep s for4 romt werec

2

thef eu 15)w owno reiA pyl

thanol, 4.17 mM EDTA–Na4, 0.83 mg/ml bovine serulbumin (BSA), 0.42 mM 3-[4,5-dimethylthiazol-2-yl]-2,iphenyltetrazolium bromide (MTT), 1.66 mM phenazthosulfate). Reaction was started with addition of 2 Uohol dehydrogenase. Plates were put in the dark at 3◦Cor 30 min. It was stopped by addition 30�l iodoacetic acid2 mM. Absorbance was read at 550 nm, and concentraf NAD+ were determined by comparison with a NAD+ stan-ard curve.

.7. DNA determination

DNA concentration was determined using the Hoeschethod[30,31]. Hoescht dye #33258 (Molecular Probe,ene, OR) was diluted at 100 ng/ml in TEN buffer (100 maCl, 10 mM EDTA, 10 mM Tris, pH 7.0) and kept in tark. The assay was made in a 96-microwell assayy mixing 100�l of working solution and 5�l of sam-le. Fluorescence was measured at 340 nm excitation60 nm emission wavelengths. DNA concentration was

ermined according to a DNA standard curve ranging froo 500 ng/�l.

.8. Western blot analysis

For poly(ADP-ribose) analysis in irradiated wor120 Gy), extracts were obtained using freshly washed wesuspended in 1× PBS and passed twice at 8000 psi irench pressure cell maintained at 4◦C (SLM-AMINCO®;ochester, NY, U.S.A.). The protein concentration was

.9. Worm treatment with PARP inhibitors

L4 larva were transferred into a 96-microwell assay p5 larvae/well), containing 100�l S medium, 50�l of Es-herichia coliNA22 strain (A550 nm 0.200), and 1.5�l ofARP inhibitors (PJ34, DHQ, and 3AB). Inhibitors wdded to the liquid medium from 100× concentrated stocolutions. Stock solutions for DHQ were made in DMSnd final concentration of DMSO in medium was 1%. Woere soaked under agitation (200 rpm) for 2 h at roomerature before irradiation.

.10. Embryonic survival determination

Sensitivity to ionizing radiation was determined ascribed by Gartner et al.[33]. After irradiation, worms werlaced individually on fresh plates and allowed to lay egg0 h. After this period, hermaphrodites were removed f

he plates, and embryos laid but not hatched after 48 hounted as dead.

.11. Bacterial cultures and extracts

The constructs pQE-PME-1 and pSD1.2 encodingull-length cDNA of pme-1and pme-2, respectively, wersed[23]. Bacteria strain M15/pREP4 (named here Mere transformed with each of the construct and grvernight at saturation at 37◦C. Fresh cultures (400 ml) we

ncubated with 5 ml overnight cultures and grown at 37◦C to6000.600. Cultures were then induced with 1 mM isopro


�-d-thiogalactoside (IPTG) at 37◦C for 4 h, except forM15 + pQE-PME-1, which was induced at 20◦C. Untrans-formed bacteria strain (M15) were also treated with 1 mMIPTG at 37◦C for 4 h. Cultures were pelleted at 3000×g for10 min at 4◦C, resuspended in 10 ml enzyme dilution buffer[100 mM Tris–HCl (pH 8.0); 100 mM MgCl2; 10% glycerol;1.5 mM dithiothreitol] and passed twice at 16,000 psi in aFrench pressure cell maintained at 4◦C (SLM-AMINCO®;Rochester, NY, U.S.A.). Extracts were centrifuged for 10 minat 1000×g at 4◦C, and the protein concentration of super-natants was determined[32]. Each supernatant was analyzedby SDS-PAGE, western blot and assayed for PARP activity.

2.12. Determination of PME-1 and PME-2 sensitivitytowards PARP inhibitors and activated DNA dependence

PARP activity was measured as described previously[23,26]. Inhibitors were added before reaction to the reactionbuffer. Samples assayed (70�l) with PARP inhibitors weremixed with 70�l of 2 × reaction buffer (200 mM Tris–HCl(pH 8.0); 20 mM MgCl2; 20% (v/v) glycerol; 3 mM dithio-threitol; 400�M NAD), 10 �l of DNase I-activated DNA(1 mg/ml) and 1�l of [ 32P]NAD (1000 Ci/mmol; 10 mCi/ml;Amersham Pharmacia Biotech). Reactions were performedfor 30 min at 30◦C and terminated with the addition of 649�lof 100 mM Tris-HCl (pH 8.0), 100�l of 3 M NaOAc (pH5.2)a for3P lvedao 04 (seea ,o a-t entp m ofa outi

w dert with2 ssaya herea CA,i sr ried,a com-p allys reu

2a

m2 ly-

acrylamide/bisacrylamide, 19.86:24) in Tris/borate/EDTA[0.09 M Tris/0.09 M boric acid/2 mM EDTA (pH 8.3)] at30 W until bromphenol blue had reached 12 cm from the ori-gin. The gel was dried and exposed[23].

3. Results

3.1. Irradiation induces pADPr production and NAD+

depletion in C. elegans

Poly(ADP-ribosyl)ation is a post-translational modifica-tion stimulated by DNA damages. This event leads, in mam-mals, to NAD+ depletion and pADPr synthesis. To determineif poly(ADP-ribosyl)ation is induced by DNA damages inC.elegans, worms were exposed to high doses of gamma rays(60 and 120 Gy). Assessment of poly(ADP-ribosyl)ation ac-tivation was done by measuring two biochemical parametersthat reflect the state of this post-translational modification:level of poly(ADP-ribose) polymers and NAD+ concentra-tion.

We noticed a significant increase in pADPr immediately(0 min) after 60 and 120 Gy irradiation treatment (2.64- and3.79-fold) (Fig. 1A). The period of time following DNA dam-age (5, 15, and 30 min) allowed the worms to recover fromtheir treatment. It also allowed the detection of pADPr syn-t ro-d ly int lyzep r isd e in-c -fold)( elo ost-i oD ac-t DPrh

sedt andw -m rra-d y,r

ac-t wasr ateds ecifica edp atedw ti-fi hicha in-t re-s t ini rred

nd 700�l of propan-2-ol. Samples were kept on ice0 min and centrifuged at 16,000×g for 10 min at 4◦C [26].ellets were washed twice with 100% ethanol and dissot 60◦C in 1 ml of 1 M KOH, 50 mM EDTA for 1 h. The pHf samples was adjusted to 9.0 with the addition of 20�lN HCl. Then, samples were applied to a DHBB resinbove). The dry eluates were dissolved in 40�l sample bufferf which 2�l were analyzed for radioactivity determin

ion on a liquid scintillation counter (Winspectral). PercADPr synthesis was calculated dividing the number cpsample with inhibitor by number cpm of a sample with

nhibitor and multiply by 100.For DNA dependence, the samples (70�l) were treated

ith 60 U DNase I for 10 min at room temperature in oro remove bacterial genomic DNA. DNase I was stopped5 mM EDTA. Samples were then submitted to a PARP as described above with the addition of activated DNA wppropriate. The reaction was stopped with 20% (w/v) T

nsoluble radioactive material (32P-poly(ADP-ribose)) waetained on glass fiber filters, washed with ethanol, air-dnd counted. Samples with activated DNA added wereared with samples without activated DNA for statisticignificant differences by ANOVA factorial. The softwased was StatView v4.51 from Abacus Concept.

.13. Poly(ADP-ribose) polymers polyacrylamide gelnalysis

The residual pellets were electrophoresed on a 20 c×0 cm× 0.15 cm 20% (w/v) polyacrylamide gel (po

hesized following DNA damage. The peak of pADPr puction detected 0 min after irradiation, decreases rapid

ime, suggesting the action of enzymes able to hydroADPr in the worm. However, a second wave of pADPetected 15 and 30 min following treatment at 120 Gy. Threases at these time points are extreme (6.86- and 7.35Fig. 1A). Treatment with 60 Gy did not induce high levf pADPr, but a significant increase occurs at 30 min p

rradiation. Therefore, it is clear thatC. elegansresponds tNA damage by stimulating a poly(ADP-ribosyl)ation re

ion similar to mammals. However, the mechanism of pAydrolysis seems to be less efficient in the worm.

The level of NAD+ drops sharply after worms are expoo 120 Gy whereas a dose of 60 Gy induces a slowereaker response (Fig. 1B). Pools of NAD+ are back to noral level and beyond after 15 min recovery from 120 Gy iiation. The reconstitution of NAD+ pools is slower at 60 Geaching original level 30 min after irradiation (Fig. 1B).

Poly(ADP-ribosyl)ation in the worm was further charerized and detection of poly(ADP-ribosyl)ated proteinsealized. Worm extracts from non-irradiated and irradipecimens were subjected to western analysis using a spnti-pADPr antibody (LP96-10). Poly(ADP-ribosyl)atroteins were detected only in worms that have been treith gamma rays (Fig. 1C). The spectrum of positively idened modified proteins ranges from about 26–212 kDa wlso characterizes poly(ADP-ribosyl)ated proteins. The

ensity pattern of modified protein reflects the biphasicponse obtained earlier while analyzing pADPr contenrradiated worms. However, it seems that a shift occu


Fig. 1. Ionizing radiations induce a typical poly(ADP-ribosyl)ation response inC. elegans. Two biochemical parameters were measured: pADPr polymerscontent and NAD+ concentration in gamma-irradiated worms. Worms (N2) were irradiated at 60 or 120 Gy and frozen in liquid Nitrogen 0, 5, 15, and 30 minafter irradiation. (A) pADPr fold inductions were determined by using the pADPr content per mg DNA of the NT sample as reference. (B) NAD+ variationswere determined by using the content in NAD+ per mg DNA of the NT sample as reference. Data were performed in triplicate. (C) Qualitative assessment ofpoly(ADP-ribosyl)ated worms proteins after irradiation at 120 Gy. Proteins (400�g) from irradiated worms taken at indicated time intervals were loaded on aSDS-PAGE and transferred onto nitrocellulose membrane. Western blot was done using specific anti-pADPr antibodies (LP-9610). Smear starting from 23 up to212 kDa represents poly(ADP-ribosyl)ated proteins. Ctrl, poly(ADP-ribosyl)ated bacterial proteins used as positive control; NT, non-treated. (D) pADPr fromC. elegansare hydrolysable by bovine poly(ADP-ribose) glycohydrolase (PARG). Each dot contains 500 fmoles of pADPr from bovine source and 570 fmolesfrom nematode source.

in time because poly(ADP-ribosyl)ated proteins are mostlymodified 5 min post-irradiation instead of 0 min in pADPrdetermination. It is also the case for the second wave of mod-ified proteins that appears 30 min after treatment (Fig. 1C).

Poly(ADP-ribose) polymers synthesized by worms treatedwith gamma rays were further characterized by digestion withthe bovine poly(ADP-ribose) glycohydrolase (PARG) in con-ditions suitable for enzymatic hydrolysis. A subsequent de-tection using specific anti-pADPr antibody failed to detectsignificant amount of pADPr from digested sample (Fig. 1D).This strongly validates our worm’s pADPr analysis, and sug-gests that the structure of pADPr found in worms is not sig-nificantly different from mammalian pADPr. Therefore, thenematodeC. elegansreacts much the same as mammals do, inregard to poly(ADP-ribosyl)ation response to DNA damage.

3.2. PARP chemical inhibitors confer radiosensitizationto C. elegans progeny

Poly(ADP-ribosyl)ation is part of the cellular response toDNA damage and DNA damage signaling. It is also involved,

in mammals, in DNA repair. Recently, it has been foundthat poly(ADP-ribosyl)ation is conserved inC. elegans[23].Therefore, we hypothesized that its abrogation in a genotoxiccontext would have deleterious effects in worms. If indeedpoly(ADP-ribosyl)ation helps DNA repair and genome sta-bility, conversely its absence would ultimately lead to deathof the worm. In order to inhibit poly(ADP-ribosyl)ation inworms, we used three well-known mammalian PARP in-hibitors namely: 3AB, DHQ, and PJ34[34]. After 2 h ofgrowth in presence of inhibitors, worms were irradiated at120 Gy and the survival of the progeny was determined.Therefore, in absence of poly(ADP-ribosyl)ation, DNA re-pair in developing embryos will be impaired and mutation ordeletion will be carried further in successive cell divisions.Cell death occurs when some critical cellular componentsbecome non-functional.

All of the PARP inhibitors tested conferred a dose-dependent decrease in embryonic survival following DNAdamage (Fig. 2). PARP inhibitors did not affect significantlyembryonic survival in absence of radiations as it stays be-tween 97 and 100%. The inhibitor 3AB was a weak sensitizer


Fig. 2. Mammalian PARP inhibitors confer radiosensitivity toC. elegansprogeny. L4 larvae were grown for 2 h at room temperature in presence of selectedPARP inhibitors followed by irradiation at 120 Gy. Larvae were then transferred to fresh plate and allowed to lay eggs for 40 h. Survival rate of the F1 generationwas determined as described in Section2. The PARPs inhibitors used were (A) 3-aminobenzamide (3AB); (B) 1,5-dihydroxyisoquinoline (DHQ); (C)N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide·HCl (PJ34); (D) levels of poly(ADP-ribosyl)ated proteins from worms treated with inhibitorsdecreased after radiation treatment. Worms were exposed to gamma rays (120 Gy) in absence (w/o inhib) or presence of PJ34 (100�M), DHQ (100�M), and3AB (1000�M). Western blot using an anti-pADPr antibody was done as described in Section2.8. Intensity of the positively identified proteins is expressedas integrated optical density following densitometric analysis.

when used at concentrations ranging from 1 to 1000�M(Fig. 2B). It correlates well with its mammalian IC50 [34].At the highest concentration, this inhibitor was able to bringdown the embryonic survival to 65% compared to 76% whenworms were irradiated without inhibitor. Likewise, the in-hibitor DHQ alone conferred only a slight radiosensitivity toembryos (Fig. 2B). The solvent in which DHQ was dissolved,DMSO, was shown to provide a slight protection againstirradiation. This suggests that a large part of DNA damagefrom radiation is mediated via oxidative damage in wormswhere the scavenging effect of DMSO may protect DNAfrom oxygen reactive species. However, this protective effectdisappears when DHQ is used at≥1�M (Fig. 2B). DHQsensitization reaches a plateau at 10�M where survival is65%.

The strongest radiosensitization is provided by PJ34. Thiswater-soluble inhibitor has significant effect on embryonicsurvival even when used at very low concentration (100 nM)(Fig. 2C). This effect is further increased at higher concen-tration where it reaches 31% survival at 100�M.

A corollary of this is poly(ADP-ribosyl)ation of target pro-tein in the worm should be reduced. In order to determinethe level of poly(ADP-ribosyl)ated proteins in worms treatedwith PARP inhibitors and irradiated, we analyzed total wormextract using an anti-pADPr antibody in a western blot (see

Section2.8). Indeed, the levels of pADPr on proteins werelower in worm treated with DHQ and PJ34 inhibitors com-pared to untreated worms (Fig. 2D). However, worm treatedwith 3AB did not show a drop in poly(ADP-ribose)-modifiedproteins. This may be due to the sensitivity of the techniqueused to detect the modified proteins. It may also be due tothe weakness of this inhibitor on endogenous PME-1 andPME-2. Taken together these data suggest that poly(ADP-ribosyl)ation inC.elegansis essential for recovery from DNAdamage.

3.3. PARP inhibitors decrease PME-1 and PME-2poly(ADP-ribosyl)ation activity

Poly(ADP-ribose) synthesis is mediated inC. elegansbymembers of thepoly(ADP-ribose)metabolismenzyme (pme)family [23]. PME-1 and PME-2 are poly(ADP-ribose) poly-merases, and are homologues of mammalian PARP-1 andPARP-2/PARP-3. We hypothesized that PME-1 and PME-2 would be inhibited by mammalian PARP inhibitors, thus,providing a molecular link between radiosensitization and en-dogenous enzymes target. We set an in vitro assay in whichrecombinant enzymes 6xHis-PME-1 and 6xHis-PME-2 ex-pressed in bacteria (Fig. 3A; [23]) were tested for their sen-sitivity towards PARP inhibitors.


Fig. 3. TheC. eleganspoly(ADP-ribose) polymerases PME-1 and PME-2 are sensitive to mammalian PARP-1 inhibitors and dependent on activated DNA.(A) Bacterial expression of recombinant 6xHis-PME-1 and 6xHis-PME-2. M15 bacteria transformed with pQE-Pme1 or pSD1.2 construct were IPTG-inducedfor 4 h at 37◦C. Equal amounts of bacterial extracts were loaded on a SDS-PAGE and stained with Coomassie blue (left panel). Levels of induction and sizeof recombinant proteins were confirmed by Western blot using an anti-His tag antibody (right panel). Arrows indicate location of 6xHis-PME-1 (111 kDa)and 6xHis-PME-2 (65 kDa) recombinant proteins. Bacterial extracts containing recombinant proteins were assayed for their PARP activity in absenceorpresence of PARP inhibitors at the indicated concentrations; (B) 3-aminobenzamide (3AB); (C) 1,5-dihydroxyisoquinoline (DHQ); (D)N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide·HCl (PJ34); (E) 6xHis-PME-1 and 6xHis-PME-2 showed an increase in activity in presence of activated DNAin a PARP in vitro assay using bacterial extract expressing the recombinant proteins. Results (mean± standard deviation) are from four determinations. Theasterisk (*) indicates a significant difference for 6xHis-PME-1 (P< 0.0021) and (P< 0.0076) for 6xHis-PME-2.

The three inhibitors used, 3AB, DHQ and PJ34, success-fully inhibited recombinant 6xHis-PME-1 and 6xHis-PME-2 in a dose-dependent manner (Fig. 3B–D). RecombinantPME-2 was almost entirely inhibited (94%) by 1 mM 3ABwhereas PME-1 was inhibited by 56% (Fig. 3B). The in-hibitor DHQ was more effective on PME-2, reaching 66%

inhibition at 100�M, and even more effective on PME-1with 80% inhibition. The solvent DMSO seems to enhancePARP activity of both PME-1 (102%) and PME-2 (132%). Asimilar effect on embryonic survival was also detected whenDMSO was used on worms (Fig. 2B). Inhibition pattern ofPME-2 with PJ34 is similar to pattern obtained with 3AB


(Fig. 3D). In both cases, the slope of inhibition is constant atrelatively low inhibitors concentrations (0–10 and 100�M).However, high inhibitors concentrations (100 and 1000�M)shift the slope at a steep angle. PJ34 is also very effectiveon PME-2 with 97% inhibition at 100�M whereas PME-1is inhibited at 81%.

Mammalian counterparts of PME-1 and PME-2, namely,PARP-1 and PARP-2, are dependent on broken DNA tostimulate their enzymatic activity[2,15]. In order to deter-mine if PME-1 and PME-2 have the same characteristic weperformed a standard PARP assay in presence or absence ofactivated DNA. Bacterial extract expressing 6xHis-PME-1and 6xHis-PME-2 were used and both showed a significantincrease in their PARP activity when activated DNA wasintroduced in the assay (Fig. 3E). The stimulation ofpoly(ADP-ribosyl)ation measured in our assay is well withinwhat mammalian PARPs are able to achieve. This may be dueto the fact that recombinant proteins used in the assay, 6xHis-PME-1 and 6xHis-PME-2, were not purified. Nevertheless,it is still possible that PARPs inC. eleganshave a weaker re-sponse to broken DNA and may be physiologically relevant.

Poly(ADP-ribose) polymers synthesized during theseassays were analyzed on polyacrylamide gel electrophoresis(Fig. 4A and B). The overall pattern follows closely thedata obtained above by scintillation counting where quantityof the pADPr is inversely proportional to the inhibitorsc izedb les.I es hesed nousP thed

4

arst s de-v neu-r n tob andD kt int naryc NAd amea

m’sN tioni ationa nset sere mu-l se)p ilar

Fig. 4. PME-1 and PME-2 chemical inhibition affects mainly poly(ADP-ribose) polymers elongation. Size distribution of poly(ADP-ribose) poly-mers synthesized by (A) 6xHis-PME-1 or (B) 6xHis-PME-2. Small pADPrpolymers are still detectable at high inhibitor concentrations whereas longpolymers are barely present.

to the structure of mammalian PARP-1[23]. Therefore, itmay bind the DNA breaks via its zinc finger motifs, exactlythe way PARP-1 binds to DNA strand breaks thereby in-ducing a boost in enzymatic activity. However, the secondzinc finger of PME-1 is significantly shorter than the onefound in mammals[23]. This feature may alter the way PME-1 interacts with DNA. It is also possible that PME-1, likePARP-1, is the enzyme that synthesizes most of the polymers

oncentration. Qualitatively, pADPr polymers synthesy PME-1 and PME-2 have similar electrophoretic profi

nhibition of poly(ADP-ribosyl)ation affects mainly thynthesis of long polymers. Therefore, taken together tata suggest a link between the inhibition of endogeARPs in the worm, namely PME-1 and PME-2, andecreased embryonic survival rate of irradiated worms.

. Discussion

The nematodeC. eleganshas been used for many yeo decipher many important biological processes such aelopment, programmed cell death, sex determination,ological networking, etc. Recently, the worm was showe an excellent model to study DNA damage responseNA damage repair processes[22]. Therefore, we undertoo

he functional analysis of poly(ADP-ribose) metabolismhe worm. The results presented here reveal the evolutioonservation of poly(ADP-ribosyl)ation in response to Damage. It shows that PME-1 and PME-2 act quite the ss their mammalian counterpart in a genotoxic context.

When DNA damage occurs after irradiation, the worAD+ content drops and the poly(ADP-ribose) concentra

ncreases. This event was predictable since PARP activnd poly(ADP-ribosyl)ation are part of early cell respo

o IR in mouse[35]. It suggests that PME-1, and to a lesxtend PME-2, will detect DNA strand breaks and sti

ate their enzymatic activities to enhance poly(ADP-riboolymer synthesis. The structure of PME-1 is very sim


Fig. 5. Model of poly(ADP-ribose) polymerases-dependent radiosensitivity inC. elegans. Ionizing radiations generate DNA damages including double strandbreaks (DSB) in genomic DNA. DSB may be detected by PME-1 (PARP-1 orthologue) through its zinc fingers and by PME-2 (PARP-2/PARP-3 orthologue)inducing poly(ADP-ribosyl)ation. Poly(ADP-ribosyl)ation of PME-1, PME-2, and surrounding nuclear proteins take place. This event may promote theactivation of signaling pathways, leading to activation of DNA repair protein complexes. It also provokes the desorption of automodified PME-1 and PME-2from DNA breaks. Poly(ADP-ribose) polymers are rapidly hydrolyzed by endogenous PARG (PME-3 and PME-4). The chemical inhibition of PME-1 andPME-2 decreases their capacity to desorb from DNA breaks, hindering the repair and normal metabolism of genomic DNA.


following DNA damage[9]. Our data suggest that PME-2also contributes to the DNA damage-dependent poly(ADP-ribosyl)ation. This would imply a redundant function withPME-1. This situation is similar to the one found in mice[21]. Therefore, the functional redundancy of these enzymesmay be physiologically very important because of its evolu-tionary conservation.

Poly(ADP-ribosyl)ation induced by ionizing radiationin the worm displays a biphasic curve (Fig. 1). The initialdecrease of the first phase is most probably due to the actionof endogenous PARG. Two PARGs are found inC. elegans,PME-3, and PME-4, and they both are able to hydrolyzepoly(ADP-ribose) polymers the same way mammalianPARG does[36,37]. PME-3 and PME-4 would thus be ableto cleave most of the polymers that modify PME-1 andPME-2, allowing them to regain their full enzyme activity.

4.1. Inhibition of PARPs leads to embryonic lethalityafter irradiation in C. elegans

We also made use of PARP inhibitors in order to demon-strate a physiological relevance to poly(ADP-ribosyl)ation inthe worm. Inhibitors 3-aminobenzamide and DHQ sensitizemammalian cells to radiotherapy[34,38]. PJ34 is a very po-tent PARP inhibitor used particularly for its protective effectagainst cell death after stroke, myocardial infarction or reoxy-g ovee ents.A m’sp J34,a E-1 e oft ARPsa alsom E-2 ct,P pairp thew

ry-oi trandb blyd vateir ayp esisic NAr aysmC ac-t bitr onp eredt r, it

prevents the desorption of PME-1 from the break. Therefore,PME-1 and PME-2 may be considered as survival factorsfor C. elegansprogeny after irradiation[40]. It is alsoimportant to mention thatpme-1was shown to protect thegenome ofC. elegansfrom deleterious mutations in normalculture condition[41]. This suggests that basal poly(ADP-ribosyl)ation from PME-1 critically regulates DNA repair ina non-genotoxic context, and that poly(ADP-ribosyl)ationadapts its regulation according to environmental conditions.

Therefore, PME-1/PME-2 act very similarly to PARP-1.In mammals, PARP-1 modifies target proteins and recruitsDNA repair proteins to the DNA damages sites, thereby pro-moting cell survival. Absence of PARP-1 may lead to celldeath after irradiation. PARP-1−/− cells are more sensitive toIR than wild type cells[40,42]. This is also true for PARP-2,which is involved in DNA damages recognition and signalingleading to DNA repair and cell survival[21]. PARP activityis even essential for proper cell development, since PARP-1and PARP-2 double mutant mouse are not viable[43].

4.2. PME-1/PME-2 in the DNA damage responsepathway in the nematode C. elegans

The DNA damage response pathway in the nematodeC.elegansis simpler than the mammalian pathway, for it im-plicates much less proteins. The known damage sensors areH tf ar-r ist.I cellcm ino forD ens-a butn reg-u oteD

P-r alinga ,i aksa dedf thew andD wert com-p ls.

A

)ff thea This

enation injuries[39]. PJ34 has never been used to imprffects of chemo- and radiotherapy for cancer treatmll of the inhibitors tested decreased survival of the worrogeny. The most potent mammalian PARP inhibitor, Plso displayed a very potent enzymatic inhibition on PMand PME-2 as well as on the embryonic survival rat

he worm. These data suggest that endogenous worm Pre very likely working as mammalian PARPs do. Theyean that poly(ADP-ribosyl)ation from PME-1 and PMis very important for the survival of the progeny. In faME-1 and PME-2 seems to be involved in the DNA rerocess that follows treatment with ionizing radiation inorm.A possible model of PME-1/PME-2-dependent emb

nic survival inC. eleganswould be as follow (Fig. 5):onizing radiations generate, among others, double sreaks (DSB) in genomic DNA. DSB are presumaetected by PME-1 through its zinc fingers which acti

ts poly(ADP-ribose) polymerase activity[23]. Poly(ADP-ibosyl)ation of PME-1 and critical nuclear proteins mromote activation of signaling pathways. PADPr synth

s a signal for recognition of DSB site[14]. PME-1/PME-2ould also interact with checkpoint proteins or activate Depair protein complexes. Activation of these pathway lead to DNA repair and cell survival or death (Fig. 5).hemical inhibition of PME-1/PME-2 prevents the inter

ion with NAD+ for pADPr synthesis but does not prohiecognition of DSB. The lack of poly(ADP-ribosyl)atirevents the proper signaling that would have gath

he repair enzymes at the site of damage. Moreove

US-1[44], MRT-2 [45], HPR-9 and RAD-5[46]. They acollowing DNA damage to induce apoptosis or cell cycleest[47]. PME-1/PME-2 may very well be added to this ln mammals, PARP-1 is a positive regulator of p53 forycle arrest and DNA repair in G1 following IR[48]. PME-1ay also interact withC. elegansp53 homologue, CEP-1,rder to promote embryonic survival. CEP-1 is requiredNA damage-induced programmed cell death but is dispble for cell cycle arrest. PME-1 may interact with CEP-1ot to provide cell cycle arrest. PME-1 could be a CEP-1lator for activation of the programmed cell death to promNA repair and cell survival.Our data strongly support the notion that poly(AD

ibose) polymerases are important in DNA damage signnd in DNA repair in the nematodeC. elegans. Moreover

onizing radiations are known to induce DNA strand brend our data suggest that poly(ADP-ribosyl)ation is nee

or the proper repair of such damage. Study of PARP inorm, where component of the DNA damage responseNA repair pathway are not numerous may provide ans

o many questions raised in human because of the highlexity and redundancy of these components in mamma

cknowledgments

We thank TheCaenorhabditisGenetics Center (CGCor providing theC. elegansN2 strain, Dr. Luc Vallieresor access to his irradiator and Dr. Guy Poirier fornti-pADPr antibody (LP-9610) among other reagents.


work is supported by a grant from the Natural Sciences andEngineering Research Council of Canada (218712-99) andby The Foundation for Research into Children’s Diseases.S.D. is a Junior 2 Research Fellow from the Fonds de laRecherche en Sante du Quebec.

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