Poly(ADP-ribo)Polymera-2(PARP-2)Is Required for Efficient Ba Excision DNA Repair in Association with PARP-1and XRCC1*
Received for publication,March 12,2002,and in revid form,April 8,2002Published,JBC Papers in Press,April 10,2002,DOI 10.1074/jbc.M202390200
Vale ´rie Schreiber‡,Jean-Christophe Ame ´‡,Pascal Dolle ´§,Ine `s Schultz‡,Bruno Rinaldi‡,Vale ´rie Fraulob§,Josiane Me ´nissier-de Murcia‡,and Gilbert de Murcia‡¶
From the ‡UPR 9003du Centre National de la Recherche Scientifique,Laboratoire conventionne ´avec le Commissariat a `l’Energie Atomique,Universite ´Louis Pasteur,Ecole Supe ´rieure de Biotechnologie de Strasbourg,boulevard Se ´bastien Brant,F-67400Illkirch and the §Institut de Ge ´ne ´tique et de Biologie Mole ´culaire et Cellulaire,CNRS/INSERM/ULP,Colle `ge de France,BP 163,67404Illkirch cedex,France
The DNA damage dependence of poly(ADP-ribo)po-lymera-2(PARP-2)activity is suggestive of its impli-cation in genome surveillance and protection.Here we
show that the PARP-2gene,mainly expresd in actively dividing tissues follows,but to a smaller exte
nt,that of PARP-1during mou development.We found that PARP-2and PARP-1homo-and heterodimerize;the in-teracting interfaces,sites of reciprocal modification,have been mapped.PARP-2was also found to interact with three other proteins involved in the ba excision repair pathway:x-ray cross complementing factor 1(XRCC1),DNA polymera ,and DNA liga III,already known as partners of PARP-1.XRCC1negatively regu-lates PARP-2activity,as it does for PARP-1,while being a polymer acceptor for both PARP-1and PARP-2.To gain insight into the physiological role of PARP-2in respon to genotoxic stress,we developed by gene dis-ruption mice deficient in PARP-2.Following treatment by the alkylating agent N -nitroso-N -methylurea (MNU),PARP-2-deficient cells displayed an important delay in DNA strand breaks realing,similar to that obrved in PARP-1deficient cells,thus confirming that PARP-2is also an active player in ba excision repair despite its low capacity to synthesize ADP-ribo polymers.
In respon to DNA interruptions,PARP-1,1the founding member of the poly(ADP-ribo)polymera superfamily,cat-alyzes the successive covalent addition of ADP-ribo units from NAD to a limited number of nuclear acceptors to form a branched anionic polymer.PARP-1is a nuclear enzyme in-volved in the detection and signaling of DNA strand breaks introduced either directly by ionizing radiation or indirectly
following enzymatic incision of a DNA lesion (abasic sites or oxidized or alkylated bas)repaired by the ba excision re-pair (BER)pathway (e for review Ref.1).The discovery of numerous PARP-1protein partners and/or poly(ADP-ribo)acceptors involved in DNA architecture (histones H1and H2B,lamin B,and high mobility group proteins)or in DNA metab-olism (DNA replication factors,DNA repair XRCC1,transcription factors,topoisomeras,and PARP-1it-lf)has shed light onto the implication of PARP-1in the process (e for review Ref.1).
The function of PARP-1in BER has long been assumed,until direct evidence demonstrated the prence of PARP-1in the BER complex,associated to XRCC1(2,3)and DNA polymera (pol)(4).The polymer produced by PARP-1upon activation by DNA breaks triggers the recruitment of XRCC1,which shows high affinity for oligo(ADP-ribosyl)ated PARP-1(3–5).The re-quirement of PARP-1in BER was established in vivo ,becau PARP-1knock-out cells displayed a vere defect in strand breaks realing following genotoxic treatment (6,7).The pref-erential role of PARP-1in long patch BER was obrved using extracts from the PARP-1knock-out cells (4).Photoaffinity labeling experiments revealed that PARP-1binds to BER in-termediates (8).In reconstituted in vitro systems containing purified human BER enzymes,PARP-1was shown to stimulate strand displacement DNA synthesis by DNA pol ,in coopera-tion with FEN-1,leading to long patch BER (9).
The mou models in which the PARP-1gene has been knocked out (10–12)revealed the dual facets of PARP-1func-tion.In proliferative cells inflicted with sub-lethal dos of DNA damage,PARP-1as a survival factor participates in DNA damage detection and signaling,leading to cell cycle arrest and DNA repair,to avoid deleterious genetic alterations (1).On the other hand,in post-mitotic cells,massive DNA damage as obrved in pathological conditions such as cerebellar or car-diac ischemia or ptic shock and to overactivate PARP-1,triggering energy depletion that leads to cell death (e for review Ref.13).
The PARP-1knock-out mice were at the origin of the discov-ery of a new DNA damage-dependent PARP protein,named PARP-2,becau an unexpected residual poly(ADP-ribo)syn-thesis could be measured in PARP-1-deficient cells following DNA damage (14,15).In addition to PARP-2(15–17),veral other PARPs were discovered almost simultaneously,all hav-ing in common a conrved catalytic domain responsible for poly(ADP-ribo)synthesis:PARP-3(17),vPARP,a 193-kDa PARP belonging to the vault particles (18),Tankyra 1and 2,two proteins associated to the telomeric protein TRF1but also found in the Golgi or in nuclear pore complexes (19–22),and the 2,3,7,8-tetrachlorodibenzo-p -dioxin-inducible TiPARP (23).
*This work was supported by Association pour la Recherche Contre le Cancer,Ligue Nationale contr
e le Cancer et Comite ´Re ´gional,Elec-tricite ´de France,Commissariat a `l’Energie Atomique and Centre Na-tional de la Recherche Scientifique.The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked “advertiment ”in accordance with 18U.S.C.Section 1734solely to indicate this fact.
¶To whom correspondence should be addresd.Tel.:33-390-24-47-07;Fax:33-390-24-46-86;E-mail:demurcia@esbs.u-strasbg.fr.1
The abbreviations ud are:PARP,poly(ADP-ribo)polymera;3-AB,3-aminobenzamide;APE1,apurinic/apyrimidinic endonuclea 1;BER,ba excision repair;BRCT,BRCA1C-terminus;,days post-coitum;dRP,deoxyribo phosphate;EST,expresd quence tags;FEN-1,flap endonuclea-1;GFP,green fluorescence protein;GST,glutathione S -transfera;h,human;m,mou;SPR,short patch re-pair;LPR,long patch repair;MEF(s),mou embryonic fibroblast(s);MNU,N -nitroso-N -methylurea;PNK,polynucleotide kina;pol,po-lymera;XRCC1,x-ray cross complementing factor 1;E,embryonic day.
T HE J OURNAL OF B IOLOGICAL C HEMISTRY
Vol.277,No.25,Issue of June 21,pp.23028–23036,2002
©2002by The American Society for Biochemistry and Molecular Biology,Inc.Printed in U.S.A.
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PARP-1and PARP-2are the only ones reported to be DNA damage-dependent,and in Arabidopsis thaliana both PARP-1 and PARP-2genes are induced by ionizing radiation(24).The N-terminal part of mammalian PARP-2contains a nuclear location signal and a functional DNA binding domain(15) distinct from that of PARP-1(two zinc fingers).The nature of this DNA binding domain has yet to be determined.
In our attempts to further characterize PARP-2and compare its biological implication with that of PARP-1with respect to DNA damage surveillance,we discovered that the expression pattern of PARP-2and PARP-1genes follows almost the same tissue distribution.The two proteins homo-and heterodimerize and poly(ADP-ribosyl)ate each other.In addition,PARP-2was found to interact with th
ide硬盘e BER proteins XRCC1,DNA pol, and DNA liga III,all being PARP-1partners as well.XRCC1 could be heteromodified by PARP-2and was able to negatively regulate PARP-2activity as it does for PARP-1.The require-ment for PARP-2in BER was demonstrated in vivo by the COMET assay in mou embryonic cells lacking PARP-2.Our results showed that PARP-2is a component of a functional BER complex in vivo,likely through dimerization with PARP-1.This strengthens the role of PARP-2as a survival factor following genotoxic stress.
EXPERIMENTAL PROCEDURES
Plasmids—The Sma I/NotI fragment encoding full-length murine PARP-2(mPARP-2)cDNA was isolate from pVL-mPARP-2(15),and sub-cloned into Hpa I/Not I sites of the pBC vector(25)in-frame with GST,allowing the expression of GST-mPARP-2fusion protein.Trun-cated forms of mPARP-2were generated by PCR and cloned in-frame with GST in the pBC vector.The Xho I/Xho I PCR product encompassing full-length mPARP-2was ligated into the Xho I site of pEGFP-C3vector (CLONTECH,Palo Alto,CA),allowing the expression of GFP-mPARP-2.Complementary oligonucleotides encoding the FLAG epitope following a methionine were linked into the Eco RV/Eco RI sites of the pIRES-eGFP vector(CLONTECH),allowing the expression of the FLAG epitope(F).The cDNA encoding full-length human XRCC1 (hXRCC1,kindly given by K.Caldecott)was
subcloned into the EcoRI sites of pIRES-FLAG vector,allowing the expression of FLAG-tagged XRCC1(F-hXRCC1).
In Situ Hybridization—In situ hybridization was performed as de-scribed in Niederreither and Dolle´(26)on rial ctions(10m)of frozen embryos or mou adult organs discted from2-or16-week CD1 mice and frozen in OCT.A Xho I/Pst I fragment from a mou PARP-1 EST clone(AA032357,Rearch Genetics,Huntsville,AL),encoding residues337–572,was subcloned into pBluescript SK(ϩ),and antin and n mPARP-1riboprobes were produced using T3and T7RNA polymeras,respectively.The murine PARP-2probe corresponding to residues8–363is described in Ame´et al.(15).Exposure varied from4 to6weeks for PARP-1and PARP-2probes.头晕该怎么办
Immunoprecipitation,GST Pull-down,and Western Blot Analys—For immunoprecipitation of purified proteins,1g of purified hPARP-1 and/or mPARP-2(as indicated)was incubated2h at4°C with20l of F1.23monoclonal anti-PARP-1antibody and1g of bovine rum albumin in1ml of LSB(20m M Tris-HCl pH8,120m M NaCl,0.1% Nonidet P-40,0.5m M phenylmethylsulfonyl fluoride)with protea inhibitors(Complete Mini,Roche Molecular Biochemicals,Mannheim, Germany).Protein G-Sepharo beads(Amersham Biosciences,Inc.) were added,and after30-min incubation at4°C,bound immune com-plexes were washed three times with LSB buffer,and the pellet
s were resuspended in Laemmli buffer and heated3min at100°C before analysis by Western blotting.For immunoprecipitation of endogenous PARP-1from HeLa cells,cells were lyd in LSB buffer20min at4°C, scraped,and centrifuge20min at13,000rpm at4°C.After preclearing with protein G-Sepharo30min at4°C,20l of F1.23anti-PARP-1 antibody was added,and immunoprecipitation was carried on as de-scribed above.
GST-pull-down analys were performed in HeLa S3cells as de-scribed in Dantzer et al.(4).
中班幼儿年龄特点For immunodetection,blots were incubated with anti-PARP-1 (Monte1/2,500(4)),anti-PARP2(Yuc1/2,500(15)),anti-XRCC1(Roman 1/5,000(3)),anti-DNA pol(1/1,000(4)),and anti-DNA liga III(1/250, kindly given by A.Tomkinson,San Antonio,TX)polyclonal antibodies or with anti-GST(1/10,000,Institut de Ge´ne´tique et de Biologie Mo-le´culaire et Cellulaire,Illkirch,France)and anti-GFP(1/1000,Roche Molecular Biochemicals,Indianapolis,IN)monoclonal antibodies.Blots where then probed with horradish peroxida-coupled condary an-tibodies(goat anti-rabbit,1/20,000or sheep anti-mou,1/20,000, Sigma Chemical Co.,St.Louis,MO),and immunoreactivity was de-tected by enhanced chemiluminescence(PerkinElmer Life Sciences, Boston,MA).When indicated,3-AB(1m M)was added2h prior to lysis and maintained throughout all the lysis and washing steps.
Heteromodification of GST Fusion Proteins by PARP-2or PARP-1—GST pull-down assays were performed as described above,except that washes were done with HSB(20m M Tris-HCl,pH8,500m M NaCl,0.5% Nonidet P-40,0.5m M phenylmethylsulfonyl fluoride).After a last wash with activity buffer(50m M Tris-HCl,pH8,4m M MgCl
2
,0.3m M dithiothreitol),each sample was split onto three,the beads were pel-leted(volume of the pellet:Ϯ20l)and resuspended in300l of activity buffer containing either300pmol of hPARP-1,600pmol of mPARP-2,or no PARP.Reaction was started by the addition of180l of activity buffer containing DNa I-activated calf thymus DNA,and [32P]NAD.Final concentrations were0.5g of DNA,1M NAD for control,and PARP-2and0.1M for PARP-1samples.In addition,each sample contained1pmol of[32P]NAD(1000Ci/mmol).After4min at 25°C,the reaction was stopped by the addition of500l of cold HSB on ice,and beads were washed three times with HSB,resuspended in12l of Laemmli buffer,and heated for3min at100°C before analysis by Western blot.
Poly ADP-ribosylation of PARP-2and XRCC1—Purified mPARP-2 (200pmol)was incubated with1-to8-fold purified hXRCC1(3)for2 min at25°C in40l of activity buffer containing300ng of bovine ru
m albumin,5M[32P]NAD(1000Ci/mmol),and100ng of DNa I activated calf thymus DNA.Reaction was stopped by addition of15l of Laemmli buffer on ice,and reaction products were analyzed by gel electrophoresis on8%SDS-PAGE and autoradiography of the Coomas-sie Blue-stained and dried gel.
Generation and Culture of Mou Embryonic Fibroblasts—Mou embryonic fibroblasts(MEFs)were isolated by micro-disction of em-bryos at day13.5of gestation resulting from intercross between PARP-2ϩ/Ϫheterozygous mice.Each embryo was genotyped by PCR to screen for the disruption of the PARP-2allele.The generation of the mice and the genotyping PCR procedure will be described elwhere. MEFs were maintained in Dulbecco’s modified Eagle’s medium,4.5 g/liter gluco medium supplemented with10%fetal bovine rum and 0.5%gentamicin.For Western blot analysis,105cells were resuspended in Laemmli buffer and sonicated,and proteins were analyzed by West-ern blot as described above,using anti-PARP-2(Yuc)and anti-PARP-1 (Monte)polyclonal antibodies.The evaluation of residual poly(ADP-ribo)synthesis in MEF cell extracts was performed as described by Ame´et al.(15).
Single Cell Gel Electrophoresis(COMET)Assay—Passage3MEFs were thawed48h prior to harvesting on60-mm Petri dishes.The following day,cells were either mock treated or expod for30
min to N-nitroso-N-methylurea(MNU)as indicatedET assay was per-formed as described in Trucco et al.(6).Slides were dried in cold ethanol,and DNA was stained prior to scoring with2g/ml ethidium bromide.Fifty COMET per slide were obrved using a Zeiss Axioplan microscope equipped with a DP50camera(Olympus)and analyzed using the VisCOMET software(Impuls Bildanaly GmbH,Gilsching, Germany)to calculate the tail moment as defined by Olive et al.(27).
RESULTS
Tissue Distribution of PARP-1and PARP-2Transcripts dur-ing Embryogenesis and in Mou Adult Tissues—In situ hybrid-ization experiments were performed to compare the expression patterns of the PARP-1and PARP-2genes at various stages of mou development and in adult tissues.Antin probes for PARP-1and PARP-2yielded specific labeling patterns that appeared similar although not perfectly identical.During early developmental stages,both genes were expresd throughout the embryo(data not shown).Differential labeling patterns became apparent by E12.5.At that stage,both genes were expresd at high levels in the developing liver and kidney(Fig. 1,A–C).Only PARP-1,however,was found to be expresd at higher levels in the genital ridge and the spinal ganglia.The signals obrved throughout other embryonic regions for both PARP-1and PARP-2antin probes were higher than for the corresponding n probes(data not shown),indi
cating a ubiq-
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uitous moderate expression of both enzymes.
床英语怎么读At E18.5(Fig.1,D –I ),PARP-1and to a lesr extent PARP-2were preferentially expresd in the thymus and in regions of the nervous system (e below).Within the developing trunk,preferential expression of PARP-1and PARP-2persisted in the liver and became restricted to the cortical region of the kidney,the spleen,adrenal gland,and in stomach and intestinal epi-thelia (Fig.1,G –I ,and data not shown).Note that PARP-1transcripts appeared more restricted than tho of PARP-2toward the ba of the intestinal crypts (G –I ,ints ).
From E14.5to E18.5,as well as in the adult mou,both genes were expresd at the highest levels in the thymus (Fig.1,D –F and data not shown).In the adult mou,PARP-1and -2expression was particularly high in the subcapsular zone of
the
F I
G .1.Comparative in situ analysis of PARP-1and PARP-2transcript distributions in mou embryos and adults organs.Each row consists of dark-field views of PARP-1(middle )and PARP-2(right )in situ hybridization signals (white dots )on adjacent ctions,and one of the corresponding bright-field views (left )to show histological details.Sagittal ctions through the trunk region of an E12.5embryo (A –C ),the head and neck of an E18.5fetus (D –F ),and the abdominal cavity of an E18.5fetus (G –I ).The ints show an enlargement of one of the intestinal loops.Frontal ctions of an adult (16-week-old)mou brain (J –L ).Sections through the testis of a 16-week-old male (M –O ).ad ,adrenal gland;cb ,cerebellum;cg ,cranial ganglia;cx ,cortex;dg ,dentate gyrus;gr ,genital ridge;hp ,hippocampus;ht ,heart;in ,intestinal epithelium (G )or interstitial tissue (M );ki ,kidney;li ,liver;lu ,lung;ob ,olfactory bulb;sg ,spinal ganglia;st ,miniferous tubules;te ,testis;th ,thymus.
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thymus(data not shown),where immature lymphocytes prolif-erate.Expression decread as lymphocytes mature and was also found in the medulla.PARP-1,and to a lesr extend, PARP-2transcripts were detected in the white pulp of the spleen,especially in the germinal centers and in Peyer’s patches in the intestine wall(data not shown)suggesting that high levels of PARP-1and-2expression are related to prolif-eration of immature lymphocytes.
At E18.5,PARP-1was preferentially expresd in specific brain regions(e the olfactory bulb,cerebellar,and cerebral cortex in Fig.1,D–F)and in the olfactory epithelia.Expression was also higher in the cranial and spinal ganglia.PARP-2 expression appeared more homogeneous in craniofacial tissues, although it was slightly up-regulated in brain and cranial/ spinal ganglia.In the adult brain(Fig.1,J–L),both PARP-1 (28)and PARP-2genes showed high expression in neuronal cells forming the stratum granulosum of the dentate gyrus and the stratum pyramidale of the hippocampus(CA1–3).Weaker expression was detected in cells of the cerebral cortex.Only PARP-1,however,was expresd at high levels in the Purkinje cell layer of the cerebellum(data not shown).
It is in testis that the expression pattern of PARP-1and PARP-2is the most distinct.PARP-1is expresd at high levels in the miniferous tubules of the developing testis(Fig.1, G–I).Expression was particularly strong in the basal layers of the miniferous epithelium(Fig.1,M–N,and Ref.29), wh
ereas no signal was detected in the luminal layers of the miniferous epithelium indicating a down-regulation of PARP-1expression at the haploid stage of meiosis.In contrast, the PARP-2signal was weak and rather homogeneous, throughout the miniferous tubules and the interstitial tissue
(Fig.1O).
Apart from testis,the expression pattern of PARP-2rem-bles that of PARP-1except that the level of expression of PARP-2is weaker.
PARP-2and PARP-1Homo-and Heterodimerize—PARP-1is known to act as a catalytic dimer(30,31).To investigate possible homodimerization of PARP-2,extracts from HeLa cells transfected with a plasmid allowing the overexpression of mu-rine PARP-2(mPARP-2)in fusion with GST were mixed with extracts from HeLa cells transfected with a plasmid allowing the expression of either mPARP-2fud to GFP,or GFP alone (Fig.2,lanes2and6,respectively).GST fusion proteins were also generated expressing truncated versions of mPARP-2 (e Fig.2):amino acids1–69(Nt,the DNA binding domain), amino acids63–202(domain E),and amino acids203–559(F, the catalytic domain).GST-fud proteins were trapped on glutathione-Sepharo beads,and copurifying GFP-tagged mPARP-2was assd by Western blot analysis using anti-GFP antibody.Fig.2shows that PARP-2is able to homodimer-ize(lane2)through its E domain(lane4).
马铃薯的功效与作用
To further investigate the possibility that PARP-2forms heterodimers with PARP-1and to prevent any cross-reaction with PARP-2,we immunoprecipitated PARP-1from HeLa cell extracts using the F1.23-specific monoclonal antibody raid against the N-terminal part of PARP-1(32).PARP-2was co-immunoprecipitated with PARP-1(Fig.3A,lane3).A negative control using an unrelated antibody did not trap either of the two proteins(lane2).The interaction between PARP-2and PARP-1was also obrved(lane4and e Fig.5B below)in the prence of the PARP inhibitor3-aminobenzamide(3-AB),in-dicating that it occurs independently of their polymerizing activity.
The complex between PARP-1and PARP-2was reconsti-tuted in vitro using purified proteins:mPARP-2was coimmu-noprecipitated with human PARP-1(hPARP-1)using the F1.23antibody(Fig.3B,lane4)demonstrating a direct interaction between PARP-2and PARP-1.
Identification of the Domains Involved in the Association of PARP-2with PARP-1—To map the interaction domain within PARP-1,GST fusion proteins were generated expressing trun-cated versions of hPARP-1(Fig.4A):amino acids1–371(A–C, the DNA binding domain),amino acids174–366(B and C), amino acids384–524(D,encompassing the BRCT domain), amino acids572–1014(F,encompassing the catalytic domain), and amino acids525–655(region E).The fusion proteins were overexpresd in HeLa cells,and GST pull-down experi-ments were performed followe
d by Western blot analys.Co-purification of endogenous PARP-2was efficient with con-structs containing either the DNA binding domain(lane2)or the BRCT domain(lane4).The domains are tho involved in the homodimerization of PARP-1(Fig.4A and Ref.30),as well as in the binding to veral partners such as XRCC1(3),DNA pol(4),DNA liga III(Fig.4A),histones,hUbc9,and tran-scription factors such as E47,TEF-1,RXR␣,Oct-1,and YY1 (e for review Ref.1),suggesting that the DNA binding and the BRCT domains of PARP-1are interfaces for protein-protein association.
In reciprocal experiments,full-length and truncated versions of mPARP-2fud to GST were expresd in HeLa cells and affinity-purified on glutathione-Sepharo beads.Copurifica-tion of endogenous PARP-1was efficient with full-length mPARP-2and with its E domain(Fig.4B,lanes2and4, respectively).The results showed that the E domain of PARP-2is involved in both the homodimerization of PARP-2 (Fig.2)and the heterodimerization with PARP-1(Fig.4B). PARP-2and PARP-1Poly(ADP-ribosyl)ate Each Other in Vitro—The ability of PARP-1to poly(ADP-ribosyl)ate PARP-2 was evaluated.Truncated versions of mPARP-2fud to GST and expresd in HeLa cells were isolated on glutathione-Sepharo beads as described above for Fig.4B,except that
the
F IG.2.PARP-2homodimerizes.Top:schematic reprentation of mPARP-2.DBD,DNA binding domain.Bottom:Extracts from HeLa cells expressing GST(lane1)and GST-tagged mPARP-2(lanes2and6) or deletion mutants of mPARP-2(lanes3–5)were mixed with extracts from HeLa cells expressing GFP(lane6)or GFP-mPARP-2(lanes1–5). Interacting proteins were analyzed by GST-pull-down and Western blot with anti-GFP antibody(top).Blot was subquently probed with anti-GST antibody(bottom).Lane7(input):1/50of the total cell extract of HeLa cells transfected with GFP-mPARP-2.
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washing buffer ud contained 0.5M NaCl and 0.5%Nonidet P-40to prevent the interaction between endogenous PARP-1and mPARP-2(data not shown).Trapped proteins on the beads were incubated for 4min with either hPARP-1or mPARP-2or neither,in the prence of [32P]NAD (0.1M for hPARP-1and 1M for mPARP-2or control)and DNa I activated calf thymus DNA.Autoradiography revealed that hPARP-1was able to poly-(ADP-ribosyl)ate the E domain of mPARP-2(Fig.5A ,panel 3),and to a l
esr extent the DNA binding domain (panel 2).Automodification of mPARP-2was weakly detected only on the E domain (panel 3).In the prence of 3-AB,no auto-/heteromodification of the E domain of mPARP-2was obrved (panel 5),confirming that the radioactive labeling detected was due to polymer synthesis.
The reciprocal experiment showed that,in the prence of 1M [32
P]NAD,mPARP-2poly(ADP-ribosyl)ates the DNA binding do-main and the BRCT domain of hPARP-1(Fig.5B ,panels 1and 3,respectively).The domains contain most of the polymer accep-tor sites in the automodification reaction of PARP-1(Fig.5B ).The results show that PARP-1and PARP-2can associate to form homo-or heterodimers and can be reciprocally poly-(ADP-ribosyl)ated.
好玩的问题
PARP-2Belongs to a BER Complex Containing XRCC1,PARP-1,DNA pol ,and DNA Liga III —Given that PARP-1is involved in ba excision repair through its association with the scaffold protein XRCC1(2–4),we examined whether
PARP-2and XRCC1could also interact.The Western blot ud to delineate the region of PARP-2interacting with PARP-1(described in Fig.4B )was probed with the anti-XRCC1anti-body.Results showed that full-length mPARP-2(Fig.4B ,lane 2)and its E-domain (lane 4)interacted with endogenou
早餐吃什么最营养s XRCC1,demonstrating that PARP-2belongs to the BER com-plex through its interaction with XRCC1.A similar approach was ud to identify the region of human XRCC1(hXRCC1)interacting with PARP-2.Fig.6A shows that only the GST fusion proteins harboring the central BRCT domain (BRCT1)of human XRCC1(lanes 3and 4)could interact with endogenous PARP-2.Neither the cond BRCT of hXRCC1(BRCT2)nor the N-terminal part of hXRCC1known to interact with DNA pol

F I
G .3.PARP-2interacts with PARP-1in vitro and in vivo .A ,co-immunoprecipitation of PARP-2with PARP-1in HeLa cell extracts.Extracts from untreated (lanes 2and 3)or 1m M 3-AB treated (lane 4)HeLa cells were incubated with the F1.23mou monoclonal anti-PARP-1antibody (lanes 3and 4)or with anti -galactosida antibody (lane 2).Bound immune complexes were analyzed by Western blot with a mixture of anti-PARP-1and anti-PARP-2polyclonal antibodies.Lane 1,control immunoprecipitation without HeLa extract.B ,coimmunopre-cipitation of purified mPARP-2with purified hPARP-1.1g of hPARP-1(lanes 1,2,and 4)was incubated without (lanes 2and 3)or with 1g of mPARP-2(lanes 1and 4)and with either F1.23anti PARP-1(lanes 2,3,and 4)or with anti-lamin (lane 1)monoclonal antibodies.Bound immune complexes were analyzed by Western blot as in A .Inputs :hPARP-1(40ng)and mPARP-2(20
ng).
F I
G .4.Interaction between PARP-2and PARP-1:mapping of the interface domains.A ,schematic reprentation of hPARP-1.GST (A and B ,lane 1)and GST-tagged deletion mutants of hPARP-1(A ,lanes 2–6)or mPARP-2(B ,lanes 2–5)were expresd in HeLa cells and interacting endogenous proteins were extracted by GST-pull-down and analyzed by Western blot,using the indicated antibodies.Blots were subquently probed with anti-GST antibody (A and B ,bottom :one reprentative GST immunodetection).A ,lane 7and B ,lane 6:crude extract of 4ϫ105HeLa cells.In panel A ,the stars show the immuno-detection of PARP-2that was carried out before GST immunodetection.
PARP-2,a Ba Excision Repair Protein
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