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Identification and Biochemical Characterization of a

Werner’s Syndrome Protein Complex with Ku70/80and Poly(ADP-ribose)Polymerase-1*

Received for publication,October 22,2003,and in revised form,January 20,2004Published,JBC Papers in Press,January 20,2004,DOI 10.1074/jbc.M311606200

Baomin Li?,Sonia Navarro?,Noriyuki Kasahara§,and Lucio Comai??

From the Departments of ?Molecular Microbiology and Immunology and §Pathology and Institute of Genetic Medicine,Keck School of Medicine,University of Southern California,Los Angeles,California 90033

Werner’s syndrome (WS)is an inherited disease char-acterized by genomic instability and premature aging.The WS gene encodes a protein (WRN)with helicase and exonuclease activities.We have previously reported that WRN interacts with Ku70/80and this interaction strongly stimulates WRN exonuclease activity.To gain further insight on the function of WRN and its relation-ship with the Ku heterodimer,we established a cell line expressing tagged WRN H ,a WRN point mutant lacking helicase activity,and used affinity purification,immu-noblot analysis and mass spectroscopy to identify WRN-associated proteins.To this end,we identified three pro-teins that are stably associated with WRN in nuclear extracts.Two of these proteins,Ku70and Ku80,were identified by immunoblot analysis.The third polypep-tide,which was identified by mass spectrometry analy-sis,is identical to poly(ADP-ribose)polymerase-1(PARP-1),a 113-kDa enzyme that functions as a sensor of DNA damage.Biochemical fractionation studies and immu-noprecipitation assays and studies confirmed that en-dogenous WRN is associated with subpopulations of PARP-1and Ku70/80in the cell.Protein interaction as-says with purified proteins further indicated that PARP-1binds directly to WRN and assembles in a com-plex with WRN and Ku70/80.In the presence of DNA and NAD ?,PARP-1poly(ADP-ribosyl)ates itself and Ku70/80but not WRN,and gel-shift assays showed that poly-(ADP-ribosyl)ation of Ku70/80decreases the DNA-bind-ing affinity of this factor.Significantly,(ADP-ribosyl)a-tion of Ku70/80reduces the ability of this factor to stimulate WRN exonuclease,suggesting that covalent modification of Ku70/80by PARP-1may play a role in the regulation of the exonucleolytic activity of WRN.

Werner’s syndrome (WS)1is a human genetic disease with many features of premature aging (1,2).The first signs of this disorder appear soon after puberty,with the symptoms becom-ing fully evident in individuals between 20and 30years old.Individuals with WS display a high incidence of diseases asso-ciated with normal aging,including atherosclerosis,osteoporo-sis,type II diabetes mellitus,and cancer.Myocardial infarction

and cancer are the most common causes of death among WS patients.The median age of death is ?47years (1,3).Cells isolated from WS patients show genomic instability and a shorter replicative life span (4).The genomic instability is characterized by an elevated rate of chromosomal transloca-tions and extensive genomic deletions (5).These findings sug-gest that genomic instability underlies the development of the diseases associated with WS.Cultured cells from WS patients are also hypersensitive to some DNA damaging agents (4),suggestive of a defect in the repair of specific DNA lesions.Werner’s syndrome is caused by mutations within a single gene,which is located on chromosome 8(6).The cDNA encodes a protein (Werner’s syndrome protein,WRN)with strong ho-mology to a class of enzymes called RecQ helicases (7).In addition,the amino-terminal region of WRN is highly homolo-gous to the nuclease domain of Escherichia coli DNA polymer-ase I and ribonuclease D (8).Helicase and exonuclease activi-ties with a 3?to 5?directionality have been demonstrated in vitro using recombinant WRN (9–14).A nuclear localization signal is found near the carboxyl-terminal end of WRN (4).All of the WRN mutations in individuals with Werner’s syndrome result in non-sense mutations or frameshifts leading to trun-cated proteins.The prevailing hypothesis is that the aberrant proteins do not enter the nucleus and are rapidly degraded.Consistent with this idea,cell lines from WS patients show no detectable WRN polypeptide (15).

A number of studies have indicated that WRN binds to proteins that are involved in DNA replication and repair,such as the replication protein A,topoisomerase I,DNA polymerase ?Fen-1,p53,proliferating cell nuclear antigen,and Rad 52(11,16–22).Although some of these proteins have been shown to influence WRN catalytic activities in vitro ,the physiological significance of these interactions remains largely unknown.In previous studies,we reported that WRN binds to Ku70/80heterodimer (Ku)(23),a factor involved in the repair of double-strand DNA breaks by non-homologous end joining (reviewed in Ref.24).Remarkably,our studies showed that Ku recruits WRN to DNA ends and alters the properties of the WRN exonuclease (23,25).Other studies have also indicated that Ku70/80is required for the WRN-mediated hydrolysis of DNA molecules containing lesions mimicking oxidative DNA dam-age (26).A functional interaction between WRN and Ku70/80is also supported by genetic studies showing that Ku80-null mice display genomic instability and shortened life span (27–31).Thus,biochemical and genetic evidence suggest that Ku80and WRN may function together in a DNA repair pathway required for the maintenance of genome integrity.

In this study,we report the identification of a cellular WRN complex composed of WRN,Ku70/80,and poly(ADP-ribose)polymerase-1(PARP-1).PARP-1is nuclear factor implicated in

*The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked “advertisement ”in accordance with 18U.S.C.Section 1734solely to indicate this fact.

?To whom correspondence should be addressed.Tel.:323-442-3950;Fax:323-442-1721;E-mail:comai@https://www.wendangku.net/doc/5e14490817.html,.1

The abbreviations used are:WS,Werner’s syndrome;WRN,Wern-er’s syndrome protein;Ku,Ku70/80heterodimer;PARP-1,poly(ADP-ribose)polymerase-1;IRES-GFP,internal ribosome entry site-green fluorescent protein.

T HE J OURNAL OF B IOLOGICAL C HEMISTRY

Vol.279,No.14,Issue of April 2,pp.13659–13667,2004

?2004by The American Society for Biochemistry and Molecular Biology,Inc.

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the control of genomic stability and mammalian life span (32,33).Our results indicate that a subpopulation of PARP-1co-elutes over ion-exchange and gel-filtration chromatography and coimmunoprecipitates with WRN and Ku70/80.Further biochemical analyses show that PARP-1poly(ADP-ribosyl)ates Ku70/80but not WRN in vitro ,and ADP-ribosylation of Ku70/80reduces its DNA-binding activity and weakens its ability to stimulate the exonuclease activity of WRN.

EXPERIMENTAL PROCEDURES

Lentiviral Vectors and Stable Cell Lines—We subcloned Flag-tagged wild type and helicase mutant (WRN H ,K577M;A 3G transition at position 1730of WRN open reading frame;Ref.34)WRN cDNAs (ex-cluding the polyadenylation signal and 3?untranslated sequences)into the polylinker sequence of a lentiviral transfer vector pRRLsin.hCMV-Puro.To generate pRRLsin.hCMV-Puro,the internal ribosome entry site-green fluorescent protein (IRES-GFP)region of the vector pRRLsin.hCMV-IRES-GFP (35)was replaced by a minimal SV40pro-moter driving the expression of the puromycin N -acetyl transferase gene.We produced recombinant lentivirus preparations by three-plas-mid transient co-transfection of human 293T cells as described by Naldini et al.(36).For lentiviral infection,293T cell cultures were trypsinized,seeded onto 60-mm plates,and incubated at 37°C for 24h.The supernatant containing viral particles was collected and added to cultures that were 30–40%confluent.After a 6-h incubation at 37°C,the supernatant was removed,the cells were washed twice and incu-bated in Dulbecco ’s modified Essential medium containing 10%serum at 37°C.Transduced cells expressing Flag-WRN and Flag-WRN H were selected in media supplemented with puromycin (10?g/ml).The ex-pression of Flag epitope-tagged proteins was analyzed by immunoblot-ting with anti-Flag antibodies (Sigma).

Construction of Plasmids and Production of Recombinant Proteins—Recombinant WRN and Ku70/80heterodimer were purified from bacu-lovirus-infected Sf9cells as described previously (23).The pVL1392Flag-PARP-1vector for expression of the recombinant Flag-PARP-1protein in Sf9cells was constructed by PCR cloning.First,the region of the open reading frame from amino acids 1–232was amplified from a PARP-1cDNA clone (Open Biosystems)by PCR using the following primers:5?-CTAGGGGCATAGGCGGAGTCTTC-3?/5?-GGGCTTTTTC-AAGCTTACTATCC-3?.The amplified fragment was digested with NdeI and HindIII and subcloned into the pcDNA3.1/HisA vector.A HindIII fragment containing the carboxyl-terminal portion of PARP-1was cloned into pcDNA3.1/HisA-Flag-PARP-1-N.The full-length PARP-1cDNA was then subcloned into the NdeI-XhoI sites of a pVL1392-Flag vector.pVL1392-Flag-PARP-1was cotransfected with linearized bacu-logold DNA (BaculoGold,Pharmingen)into Sf9cells to generate the recombinant baculovirus.For the purification of recombinant Flag-PARP-1,baculovirus-infected cells were lysed in lysis buffer (10m M Hepes,pH 7.5,100m M NaCl,1.5m M MgCl 2,0.5%Nonidet P-40),and Flag-PARP-1was purified by chromatography on DEAE-Sepharose and anti-Flag resin columns.

Purification of the WRN Complex—100mg of nuclear extracts pre-pared from cells expressing Flag-WRN H were incubated with anti-Flag beads at 4°C for one hour.After extensive washes,bound proteins were eluted with BCO buffer (1M KCl,10m M Tris HCl,pH 7.5,1m M EDTA,5%glycerol,1m M dithiothreitol,0.5m M phenylmethylsulfonyl fluoride,5?g/ml leupeptin,5?g/ml aprotinin)(23),resolved by SDS-polyacryl-amide gel electrophoresis,and visualized by silver staining.For the identification of the 120-kDa associated factor by mass spectrometry,we isolated the WRN complex from ?1.0g of nuclear extracts prepared from 293T-WRN H cells.After the immunopurification of the WRN com-plex,the proteins were separated by SDS-PAGE and stained with Coomassie blue.The gel was extensively destained,and the protein bands were excised from the gel and shipped to the Howard Hughes Medical Institute Biopolymer Facility and W.M.Keck Foundation

Biotechnology Resource Laboratory at Yale University for tryptic pep-tide digestion and sequencing by mass spectrometry.Twenty-four pep-tides matched PARP-1amino acid sequences (see Table I).

Immunoprecipitation—Nuclear extracts from 293T cells were pre-pared as described previously (23).The immunoprecipitation of cellular WRN was carried out using antibody generated by immunization of rabbits with a recombinant WRN amino-terminal fragment (amino acids 1–423).Rabbit immunization and sera production were con-tracted to an outside company (Bethyl Inc.).Immunoprecipitation prod-ucts were resolved by SDS-PAGE and transferred to nitrocellulose.Western blot analyses were performed by using antibodies raised against WRN,Ku70,and Ku80(Santa Cruz Biotechnology),PARP-1(Santa Cruz Biotechnology)and poly(ADP-ribose)(Trevigen).

Chromatographic Analysis of WRN,Ku70/80,and PARP-1—Nuclear extracts (5ml,4mg/ml)prepared according to the standard protocol of Dignam et al.(37)were loaded onto a 1-ml DEAE-Sepharose fast flow (Amersham Biosciences)column equilibrated in buffer A (20m M Tris,pH 7.5, 1.0m M EDTA,10%glycerol, 1.0m M dithiothreitol,and a protease inhibitor mixture)containing 150m M KCl.The flow-through (containing WRN,PARP-1,and Ku70/80)was then applied to a 1.0-ml SP-Sepharose fast flow (Amersham Biosciences)column,which was step-eluted in buffer A containing 0.4KCl.An equal volume (15?l)of the nuclear extract,DEAE-Sepharose flow-through,SP-Sepharose flow-through (SP-0.15),and 0.4M KCl eluate (SP-0.4)were loaded onto an SDS-8%polyacrylamide gel and analyzed by immunoblotting with WRN,Ku70,and PARP-1antibodies.The flow-through from the SP-Sepharose column (SP-0.15)was centrifuged at 100,000?g for 60min,and 1ml of the supernatant was loaded onto a Superose 6column (SP-0.15Superose,90ml).The column was run at 0.4ml/min with buffer A containing 150m M KCl,and eluates were collected in 0.6-ml fractions.Aliquots (15?l for the Ku70/80immunoblot;100?l for the WRN immunoblot)from alternate fractions were analyzed by SDS-PAGE and immunoblotted with antibodies raised against WRN and Ku70.For the analysis of WRN,100?l of the indicated fractions were subjected to trichloroacetic acid precipitation prior to loading on the SDS-polyacrylamide gel.Likewise,the 0.4M KCl eluate from the SP-Sepharose column (SP-0.4)was dialyzed in buffer A containing 0.15M KCl,centrifuged at 100,000?g for 60min,and 1ml of the supernatant was loaded onto a Superose 6column (SP-0.4Superose,90ml).The column was run as described above.Aliquots (15?l for PARP-1and Ku70/80immunoblots;100?l for WRN immunoblot)from alternate fractions were analyzed by SDS-PAGE and immunoblotted with anti-WRN,PARP-1,and Ku70antibodies.Quantitation of signal intensities was performed by densitometry scanning of x-ray films.For the immu-noprecipitation reactions,0.8ml of combined fractions 45–46and 0.8ml of combined fractions 47–48from the appropriate gel-filtration column (SP-0.15Superose 6or SP-0.4Superose 6)were incubated with 5?g of anti-WRN antibody and 15?l of protein A-Sepharose beads for 2h.The immunoprecipitated products were washed extensively,re-leased by boiling in SDS-sample buffer,resolved on an SDS-8%PAGE,and analyzed by immunoblots with antibodies raised against WRN,PARP-1,and Ku70.

Exonuclease Assay—WRN exonuclease activity was analyzed as de-scribed previously (23,25)using a 20-oligomer A1(CGCTAGCAATAT-TCTGCAGC)and a 46-oligomer A2(GCGCGGAAGCTTGGCTGCA-GAATATTGCTAGCGGGAAATCGGCGCG)partially complementary to A1.Oligonucleotide A1was labeled at the 5?end with [?32P]ATP and T4DNA kinase.The oligonucleotides were annealed by boiling and cooled gradually to room temperature.Reaction mixtures contained 40m M Tris-HCl (pH 7.5),4m M MgCl 2,5m M dithiothreitol,1m M ATP,0.1mg/ml bovine serum albumin,40fmol of DNA substrates (100,000cpm),and increasing amounts (50,100,and 200fmol)of Ku70/80or poly-(ADP-ribose)ated Ku70/80and WRN in a final volume of 10?l.The reaction mixtures were incubated at room temperature for 20min,and each reaction was terminated by the addition of 2?l of a formamide solution.After incubation at 95°C for 3min,the reaction products were

T ABLE I

PARP-1peptides identified by mass spectrometry

IEREGECQR SKLPKPVQDLIK VVDRDSEEAEIIR MIFDVESMKK LQLLEDDKENR IAPPEAPVTGYMFGK AMVEYEIDLQK QQVPSGESAILDR VVSEDFLQDVSASTK GDEVDGVDEVAK KPPLLNNADSVQAK MVDPEKPQLGMIDR SWGRVGTVIGSNK

HPDVEVDGFSELR EELGFRPEYSASQLK

GGSDDSSKDPIDVNYEK FYPLEIDYGQDEEAVK GGAAVDPDSGLEHSAHVLEK NREELGFRPEYSASQLK TGNAWHSK AEPVEVVAPR EDAIEHFMK GIYFADMVSK TLGDFAAEYAK

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resolved by 16%polyacrylamide-urea gel electrophoresis,visualized by autoradiography and phosphoimager analyzer,and quantified with Im-ageQuant software (Molecular Dynamics).

Electrophoretic Mobility Shift Assay —A 20-mer A1was labeled using [?32P]ATP and T4polynucleotide kinase and then annealed to a par-tially complementary 46-mer A2.Radiolabeled double-strand oligonu-cleotides (80fmol,200,000cpm)were incubated with increasing amounts (100–300fmol)of unmodified Ku70/80or poly(ADP-ribo-syl)ated Ku70/80in 15?l of buffer (10m M Tris-HCl,pH 7.5,100m M NaCl,0.5m M EDTA,and 10%glycerol)at 25°C for 10min.The samples were resolved by electrophoresis at 10V/cm through a 4%polyacryl-amide gel at 4°C.The gel was dried,and reaction products were visualized by autoradiography and PhosphorImager analyzer and quantified by ImageQuant software (Molecular Dynamics).

(ADP-ribosyl)ation of Proteins —20?g of purified Flag-PARP-1was incubated with 2?g His-Ku70/80or His-WRN in a reaction mixture (500?l)containing 100m M Tris pH 8.0,10m M MgCl 2,1m M dithiothre-itol,20ng/?l sonicated salmon sperm DNA,and 20?M NAD ?.After incubation at 30°C for 2h,the salts concentration in the reaction was adjusted to 400m M KCl,and the reaction mixture was incubated with anti-Flag beads at 4°C for one hour.The supernatant was collected and incubated with metal affinity resin (Talon,Clontech)at 4°C for one hour.After extensive washes with 20m M Tris,pH 8.0,150m M KCl,1m M MgCl 2,10%glycerol,and a mixture of protease inhibitors,Ku70/80or WRN were eluted with buffer containing 100m M imidazole,dialyzed,and analyzed by SDS-polyacrylamide gel electrophoresis,silver stain-ing,and immunoblotting,or snap frozen at ?80°C.

RESULTS

Isolation of WRN-associated Proteins from Cells Stably Ex-pressing Flag-WRN H —To assist in the purification of a native

WRN protein complex,we generated stable 293T cells using recombinant,replication-defective,lentivirus vectors express-ing either wild-type Flag-WRN or Flag-WRN H ,a WRN protein carrying a point mutation (K577M)that inactivates the heli-case activity (25,34).Because Flag-WRN is expressed at ex-tremely low levels (Fig.1A ,lane 1),this cell line is not partic-ularly useful for biochemical studies.One possible explanation for this effect is that the increased level of functional WRN may be toxic to the cell.On the other hand,the level of expression of Flag-WRN H (lane 2),which is comparable with that of the endogenous WRN (data not shown),is sufficient for the isola-tion and biochemical characterization of WRN H from cell ex-tracts.Thus,we prepared nuclear extracts from the 293T-WRN H cells and purified the tagged protein by affinity chromatography on anti-Flag resin.In parallel,extracts from 293T cells that were infected with a control lentivirus were subjected to the same purification procedure.Proteins bound to the affinity column were eluted with high salts and examined by SDS-polyacrylamide gel electrophoresis and silver staining.This analysis revealed the presence of three polypeptides of ?70,90,and 120kDa,respectively.These proteins were

eluted

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G .1.Identification of a WRN protein complex in cells ex-pressing Flag-WRN

H .A ,lysates were prepared from 293T cells trans-duced with lentiviruses expressing Flag-WRN (lane 1),Flag-WRN H (lane 2),or no protein (lane 3),analyzed by SDS-PAGE,and immuno-blotted with antibody against the Flag https://www.wendangku.net/doc/5e14490817.html,ne 4,purified recombi-nant Flag-WRN.B ,cell extracts prepared from a 293T cell line stably expressing Flag-WRN H ,a helicase mutant WRN (lanes 2and 4),and the parental 293T cells (lanes 1and 3)were incubated with anti-Flag beads;after extensive washes,the bound proteins were eluted from the beads with BCO buffer (see “Experimental Procedures ”).Under these conditions,the antigen (Flag-WRN H )and the nonspecific binding pro-teins remain bound to the beads,whereas the proteins associated with Flag-WRN H are eluted.Eluted proteins (lanes 1and 2)and proteins that remained bound to the beads (lanes 3and 4)were separated by SDS-8%PAGE and analyzed by silver staining.Arrow ,novel WRN-associated factor.C ,eluates from the control (lane 1)and Flag-WRN (lane 2)immunopurification reactions were resolved by SDS-8%PAGE and analyzed by immunoblotting with anti-PARP-1(top panel )and anti-Ku70and anti-Ku80(bottom panel )

antibody.

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G .2.WRN binds to PARP-1in vivo and in vitro .A ,PARP-1and Ku70/80are coimmunoprecipitated with WRN from cell extracts.Nu-clear extracts from 293T cells were subjected to immunoprecipitation with anti-WRN (lane 3)or anti-?-actin (lane 2)antibodies.Immunopre-cipitation products were analyzed by Western blotting with anti-WRN (top panel ),anti-PARP-1(middle panel ),and a mixture of anti-Ku70and anti-Ku80(bottom panel )https://www.wendangku.net/doc/5e14490817.html,ne 1contains purified re-combinant WRN,PARP-1,and Ku70/80as https://www.wendangku.net/doc/5e14490817.html,ne 4contains 10%of the extract used in the immunoprecipitation.B ,in vitro protein-protein interactions between WRN,PARP-1,and Ku70/80.Purified Flag-PARP-1(2.0?g)(lanes 6and 8)or Flag-HCV polymerase (2.0?g)(negative control:lanes 5and 7)were immobilized on anti-Flag beads and then incubated with 300?l (2.0?g)of purified His-WRN alone (lane 6)or a mixture containing purified His-WRN and Ku70/80(lane 8).After several washes,the bound proteins were solubilized in SDS-containing buffer and analyzed by immunoblotting with anti-WRN (top panel ),anti-Flag (middle panel ),and a mixture of anti-Ku70and https://www.wendangku.net/doc/5e14490817.html,nes 1–4show 5%of the protein inputs used in the protein-binding assay.

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G .3.Fractionation of WRN,PARP-1,and Ku70/80by ion-exchange and gel-filtration column chromatography.A ,graphic representation of the fractionation scheme used to analyze WRN,PARP-1,and Ku70/80complex formation.B ,nuclear extracts from 293cells were fractionated on DEAE-Sepharose and SP-Sepharose as described under “Experimental Procedures.”The nuclear extract (15?l,lane 1),DEAE flow-through (15?l,lane 2),SP-Sepharose 0.15M KCl eluate flow-through (15?l,lane 3)and 0.4M KCl eluate (15?l,lane 4)were analyzed by immunoblotting with anti-WRN,anti-PARP,and anti-Ku70antibodies.C ,the 0.15M KCl eluate (containing WRN and Ku70/80)and the 0.4M KCl eluate (containing WRN,PARP-1,and Ku70/80)from the SP-Sepharose column were independently fractionated on Superose 6columns (SP-0.15superose 6,top panel ;SP-0.4superose 6,bottom panel )as described under “Experimental Procedures.”Indicated fractions (fr.#)were analyzed by SDS-PAGE and immunoblotted with antibodies against WRN,PARP,and Ku70.Peak fractions for each factor were run on the same gel to eliminate possible differences between gels or membranes.The position of the peaks of elution of the molecular mass standards (thyroglobulin and

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specifically from the resin incubated with the 293T-WRN H nuclear extract and were absent from the eluate of the control resin (Fig.1).The 70-and 90-kDa polypeptides were identified as Ku70and Ku80,respectively,by immunoblot analysis (Fig.1C ).To identify the 120-kDa polypeptide,the protein band was excised from the SDS-polyacrylamide gel,subjected to proteo-lytic digestion,and analyzed by matrix assisted laser desorp-tion ionization mass spectrometry.Data base searches indi-cated that the 120-kDa polypeptide is identical to PARP-1(see Table I),an enzyme that is activated by DNA damage and utilizes NAD ?to catalyze the addition of poly(ADP-ribose)on target proteins.The initial identification was confirmed by immunoblot analysis with monoclonal anti-PARP-1antibody (Fig.1C ).

Physical Interaction between WRN and PARP-1in Human Cells —To provide further evidence that PARP-1interacts with endogenous WRN in vivo ,we immunoprecipitated WRN from nuclear extracts prepared from 293T cells using anti-WRN antibodies.As a control,the same nuclear extract was sub-jected to immunoprecipitation with antibody against ?-actin.The products of the immunoprecipitation reactions were re-solved by SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting.The results indicate that PARP-1coimmu-noprecipitates with WRN but not with ?-actin (Fig.2A ).The Ku70/80complex is also detected in the immunoprecipitation reaction with anti-WRN antibodies,confirming the results of the immunopurification of Flag-WRN H .Treatment of the nu-clear extract with DNase I prior to the immunoprecipitation yielded identical results (data not shown).

Previous studies have shown that PARP-1binds to Ku70/80(38,39);therefore,it is possible that Ku70/80mediates the interaction between WRN and PARP-1.To determine whether WRN binds directly to PARP-1,recombinant Flag-PARP-1was immobilized on anti-Flag beads and incubated with recombi-nant WRN (His-WRN).In parallel reactions,immobilized Flag-PARP1was incubated with Ku70/80or with a mixture contain-ing Ku70/80and WRN.After washing the beads,the bound proteins were resolved by SDS-PAGE and analyzed by Western blotting with antibodies against WRN,Ku70/80,and PARP-1.As shown in Fig.2B ,WRN binds to PARP-1in the absence of Ku70/80(lane 6),suggesting that the interaction between WRN and PARP-1in the WRN complex is mediated,at least in part,by direct interaction between these two proteins.Similarly,WRN binds to PARP-1in the presence of Ku70/80(lane 8).These results suggest that these factors can individually asso-ciate with each other and can form a trimeric complex in vitro .Analysis of the WRN Complex by Ion Exchange and Gel Filtration Chromatography —To further examine the relation-ship among WRN,Ku70/80,and PARP-1and determine whether there is evidence for the existence of a stably associ-ated complex in vivo ,we performed a three-step fractionation of the nuclear extracts using a DEAE ion-exchange column,SP-Sepharose ion-exchange column,and Superose 6gel-filtration columns (Fig.3).The flow-through of the DEAE column,which contains PARP-1,WRN,and Ku70/80as detected by immuno-blotting (Fig.3B ,lane 2),was applied to an SP-Sepharose column.Immunoblot analysis of the fractions from the step-

eluted SP-Sepharose column indicates that WRN and Ku70elute in both the flow-through (SP-0.15)(lane 3)and the 0.4M KCl fraction (SP-0.4)(lane 4),whereas PARP-1elutes in the 0.4M KCl fraction (SP-0.4)(lane 4).The SP-Sepharose flow-through (SP-0.15)and 0.4M KCl fraction (SP-0.4)were then individually fractionated further on Superose 6gel-filtration columns.The analysis of the fraction eluted from the Superose 6column loaded with the SP-Sepharose flow-through fraction (SP-0.15Superose 6)indicates that WRN and a portion of Ku coelute in fractions 44–50(Fig.3C ,top panel ).The

immunoblot

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G .4.Ku70and Ku80are poly(ADP-ribosyl)ated by PARP-1.A ,auto-poly(ADP-ribosyl)ation of PARP-1.Purified PARP-1was incu-bated with 20?M NAD ?in the absence (lane 3)or presence of increasing amounts of sonicated DNA (lanes 1and 2)or with 20ng/?l of sonicated DNA in the absence (lane 7)or presence of increasing amounts of NAD ?(lanes 4–6).Poly(ADP-ribosyl)ation of PARP-1was detected by silver staining of the gel.B ,WRN and Ku70/80were purified from insect cells infected with recombinant baculoviruses expressing His-WRN or His-Ku70and -Ku80.WRN and Ku70/80were separately incubated with PARP-1in the presence of sonicated DNA and NAD ?(WRN,lanes 2and 6;Ku70/80,lanes 4and 8)or buffer only (WRN,lanes 3and 7;Ku70/80,lanes 5and 9).WRN and Ku70/80were collected from the respective reaction mixtures by incubation with metal affinity resin,solubilized in SDS-containing buffer,resolved by SDS-PAGE,and analyzed by silver staining (lanes 2–5)and immunoblotting with anti-poly(ADP-ribo-syl)ated (?-PAR )antibody (lanes 6–9).Lane 1contains a molecular mass marker standard (HMW standard,Bio-Rad).

catalase)was determined by fractionation of a solution containing these two proteins under the same experimental conditions.Signal intensities were determined by densitometry scanning of the blots and are plotted in arbitrary units (A.U.)as a function of the fraction number.Immunoblot analyses of fractions 12–20of the SP-0.4Superose 6column and fractions 64–70of the SP-0.15Superose 6column did not reveal any detectable amounts of WRN,Ku70,and PARP-1and are not shown.Bracketed fractions represent the fractions used in the immunoprecipitation reactions shown in panel D .D ,coimmunoprecipitation of WRN,PARP-1,and Ku70/80from gel-filtration column fractions.We combined the SP-0.15Superose 6column fractions 45/46and fractions 47/48(lanes 1and 2,respectively)and the SP-0.4Superose 6column fractions 45/46and fractions 47/48(lanes 3and 4,respectively).Pooled fractions were individually incubated with anti-WRN antibody and subjected to immunoprecipitation with protein A-Sepharose.Immunoprecipitated proteins were analyzed by immunoblotting with anti-WRN (top panel ),anti-PARP-1(middle panel ),and anti-Ku70(bottom panel )antibodies.

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G .5.Poly(ADP-ribosyl)ation of Ku70/80impairs DNA binding and reduces the Ku-mediated stimulation of WRN exonuclease activity.A ,Ku70/80was purified from insect cells coinfected with recombinant baculoviruses expressing human His-Ku70and -Ku80.Ku70/80was incubated with PARP-1in the presence of sonicated DNA and NAD ?(lanes 1and 3)or buffer only (lanes 2and 4).Ku70/80were collected by incubation with a metal ion resin,solubilized in SDS-containing buffer,resolved by SDS-PAGE,and analyzed by silver staining (lanes 1and 2)and

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analysis also indicates that a minor fraction of Ku70elutes in the void volume,possibly suggesting the presence of high mo-lecular weight Ku complexes or aggregates.

A similar analysis of the fractions eluted from the Superose 6column loaded with the SP-Sepharose 0.4M KCl fraction (SP-0.4Superose 6)shows that WRN coelutes with a portion of PARP-1and Ku in fractions 44–52(Fig.3C ,bottom panel ).Note that these data are only suggestive of complex https://www.wendangku.net/doc/5e14490817.html,plex formation per se was then tested by direct immuno-precipitation of these fractions by using anti-WRN antibodies and by probing the precipitated proteins for immunoreactivity against WRN,PARP-1,and Ku70.As shown in Fig.3D ,Ku70coimmunoprecipitated with WRN from pooled fractions 45/46and fractions 47/48of the SP-0.15Superose 6column (Fig.3D ,lanes 1and 2),and Ku70and PARP-1coimmunoprecipitated with WRN from pooled fractions 45/46and fractions 47/48of the SP-0.4Superose 6column (lanes 3and 4).These results suggest that two WRN complexes may exist in vivo ,one with Ku and another with both Ku and PARP-1.

Poly(ADP-ribosyl)ation Reduces Ku70/80DNA-binding Ac-tivity —PARP-1is a nuclear protein that binds to DNA strand breaks and catalyzes ADP-ribosylation of itself and other nu-clear proteins by using NAD ?as a cofactor.This catalytic activity requires DNA.To establish that our preparation of purified recombinant PARP-1was able to poly(ADP-ribosyl)ate itself in a DNA-dependent manner,we incubated recombinant PARP-1in the absence or presence of increasing amounts of DNA and NAD ?.As shown in Fig.4A ,recombinant PARP-1becomes poly(ADP-ribosyl)ated in the presence but not in the absence of DNA,as indicated by the appearance of high molec-ular mass products in the silver-stained gel (compare Fig.4A ,lanes 1and 2with lane 3).Importantly,poly(ADP-ribosyl)ation of PARP-1depends on the presence of NAD ?,as incremental formation of high molecular mass products is observed in the presence of increasing amounts of NAD ?,and the omission of NAD ?from the reaction mixture prevents the formation of these products (Fig.4A ,lanes 4–7).To determine whether WRN and Ku70/80were substrates of the poly(ADP-ribose)polymerase activity of PARP-1,purified WRN and Ku70/80were individually incubated with PARP-1in the presence of NAD ?and DNA.WRN and Ku70/80were then immunoprecipi-tated from the respective reaction mixtures,analyzed by SDS-polyacrylamide gel electrophoresis,and immunoblotted with a monoclonal antibody that recognizes poly(ADP-ribosylation)(anti-PAR antibody).The results of this experiment indicate that PARP-1poly(ADP-ribosyl)ates Ku70/80(Fig.4B ,lane 8)but not WRN (Fig.4B ,lane 6).Interestingly,the apparent molecular mass of Ku70and Ku80does not change signifi-cantly after covalent modification,suggesting that only a few ADP-ribose molecules are added to both Ku70and Ku80.

Poly(ADP-ribosyl)ation Inhibits Ku70/80DNA-binding Ac-tivity and Reduces the Ku-dependent Stimulation of WRN Ex-onuclease Activity —The addition of ADP-ribose molecules in-creases the negative charge of a protein,which is thought to alter the DNA-binding properties of the target factor.To deter-

mine whether ADP-ribosylation influences the DNA-binding activity of Ku70/80,we purified in vitro poly(ADP-ribosyl)ated Ku70/80and unmodified Ku70/80.Ku70/80was incubated with PARP-1in the presence of DNA and NAD ?to allow poly(ADP-ribosyl)ation and purified by affinity chromatography.The pu-rity of unmodified and poly(ADP-ribosyl)ated Ku70/80was de-termined by silver staining of SDS-polyacrylamide gels (Fig.5A ,lanes 1and 2),and poly(ADP-ribosyl)ation was confirmed by immunoblot analysis with anti-PAR antibody (lanes 3and 4).Increasing amounts of unmodified or poly(ADP-ribosyl)ated Ku70/80(Fig.5A )were incubated with a radiolabeled double-strand oligomer,and DNA-binding activity was analyzed by electrophoretic mobility shift assays (Fig.5,B and C ).The results of this experiment indicate that ADP-ribosylation sig-nificantly reduced the DNA-binding activity of Ku70/80(com-pare lanes 2–4to 5–7),suggesting that this covalent modifica-tion alters the DNA-binding properties of this factor.

We have shown previously that Ku70/80recruits WRN to DNA ends and stimulates WRN exonuclease activity.Because PARP-1has been reported to bind to DNA ends (40),we exam-ined whether PARP-1may influence WRN exonuclease activ-ity.We performed WRN exonuclease assays with PARP-1in the presence and absence of Ku70/80and found that unmodi-fied PARP-1or poly(ADP-ribosyl)ated PARP-1do not affect the exonuclease activity of WRN nor alter the strong stimulation of this activity by Ku70/80(data not show).Then we wanted to determine whether poly(ADP-ribosyl)ation of Ku70/80influ-ences WRN exonuclease activity.Because the experimental conditions used for the exonuclease assay are not suitable for PARP-1poly(ADP-ribosyl)ation activity,Ku70/80was poly-(ADP-ribosyl)ated in vitro prior to its addition to the exonucle-ase reaction (Fig.5A ).WRN was then incubated with a radio-labeled double-strand oligonucleotide in exonuclease buffer in the presence of poly(ADP-ribosyl)ated Ku70/80or unmodified Ku70/80.The products of these reactions were separated by denaturing acrylamide gel electrophoresis and analyzed by autoradiography and PhosphorImager analyzer.The results of these experiments show that poly(ADP-ribosyl)ated Ku70/80leads to a modest increase in DNA hydrolysis as compared with the unmodified factor (Fig.5,panels D and E ),indicating that poly(ADP-ribosyl)ation reduces the ability of Ku70/80to stim-ulate the exonuclease activity of WRN.

DISCUSSION

In this study,we purified a WRN complex by affinity chro-matography and identified its components by immunoblot analysis and mass spectroscopy.We found that WRN resides in a complex with Ku70/80and PARP-1.Coimmunoprecipitation assays indicated that PARP-1binds to WRN and Ku70/80in vivo ,and in vitro protein-binding studies with purified factors show that the interaction between PARP-1and WRN is direct and that WRN can form a trimeric complex with PARP-1and Ku.Two groups (von Kobbe et al.and Adelfalk et al.,Refs.41and 42,respectively)have reported an interaction between WRN and PARP-1while this paper was in review.The identi-

immunoblotting with anti-poly(ADP-ribosyl)ated (?-PAR )antibody (lanes 3and 4).B ,poly(ADP-ribosyl)ated Ku70/80binds to DNA less efficiently than unmodified Ku70/80.A 32P-labeled 5?-20-mer(A1)/46-mer(A2)DNA substrate was incubated with 100–300fmol of Ku70/80(lanes 2–4)and poly(ADP-ribosyl)ated Ku70/80(lanes 5–7)at room temperature for 10min.The reactions were analyzed by 4%native polyacrylamide gel electrophoresis,and the DNA-protein complexes were visualized by autoradiography (lane 1,DNA probe only;lanes 2–4,100,200,and 300fmol of Ku70/80,respectively;lanes 5–7,100,200,and 300fmol of poly(ADP-ribosyl)ated Ku70/80,respectively).C ,the DNA-binding activity of unmodified or poly(ADP-ribosyl)ated Ku70/80(panel B )is plotted in a bar graph as a percentage of bound DNA probe.D ,poly(ADP-ribosyl)ation of Ku70/80reduces the stimulation of WRN exonuclease activity.100fmol of purified WRN and 50,100,and 200fmol of purified Ku70/80and poly(ADP-ribosyl)ated Ku70/80were incubated with a 3?-recessed,32P-labeled 5?-20-mer/46-mer DNA substrate at room temperature for 20min.Products were analyzed by 16%polyacrylamide-urea denaturing gel and autoradiography (lane 1,DNA probe only;lane 2,100fmol WRN;lanes 3–5,100fmol of WRN and 50,100,and 200fmol of Ku70/80,respectively;lanes 6–8,100fmol of WRN and 50,100,and 200fmol of poly(ADP-ribosyl)ated Ku70/80,respectively).E ,the relative WRN exonuclease activity in the presence of increasing amounts of unmodified or ADP(ribosyl)ated Ku70/80(panel D )is plotted in a bar graph as 3?nucleotides removed from the DNA substrate.

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fication of Ku70/80as one of the components of the WRN complex is consistent with previous studies(23,25,43),which established Ku70/80as a functional partner of WRN and sug-gested a possible role for WRN in DNA repair.The finding that PARP-1,a factor implicated in the cellular response to DNA damage,is a component of the WRN complex supports the idea that WRN may be involved in a pathway that monitors genome integrity.This interpretation is in agreement with a recent genetic study,which showed that double knockout mice lacking WRN and PARP-1display chromosomal instability and shorter life span(44).

PARP-1is a highly conserved eukaryotic protein that binds to single-and double-strand DNA breaks and is thought to function as a sensor of DNA damage(32,33).In response to DNA damage induced by ionizing radiation,alkylating agents, and oxidants,PARP-1binds to DNA.DNA binding causes the activation of PARP-1catalytic activity,which utilizes NAD?to catalyze the addition of multiple ADP-ribose molecules to ac-ceptor proteins.A number of studies have reported that acti-vated PARP-1poly(ADP-ribosyl)ates proteins such as histones, p53,topoisomerases,lamins,and PARP-1itself(45–50).Poly-(ADP-ribosyl)ation of p53has been reported to influence the DNA-binding activity of this tumor-suppressor protein(51). Moreover,poly(ADP-ribosyl)ation of chromosomal proteins has been proposed to alter the nucleosomal structure near the DNA strand breaks and to promote the access of repair enzymes to these sites(52).We have examined whether WRN and Ku70/80 are substrates of PARP-1enzymatic activity and have shown that PARP-1poly(ADP-ribosyl)ates Ku70and Ku80but not WRN.In addition,our analysis indicates that poly(ADP-ribo-syl)ation of Ku70/80alters the DNA-binding activity of this factor and inhibits the Ku70/80-mediated stimulation of WRN exonuclease activity.Thus,these results indicate that,through covalent modification of Ku70/80,PARP-1modulates WRN exonuclease activity.Conversely,we did not observe any sig-nificant alteration of WRN helicase activity by PARP-1in the presence or absence of Ku70/80(data not shown).Given the role of Ku70/80and PARP-1in the recognition of DNA breaks, our findings suggest that WRN,once recruited to the site of DNA damage,may participate directly in the processing and resolution of the broken DNA ends.Because there are several-fold more Ku and PARP-1molecules than WRN in a cell,it is conceivable that WRN in the context of this complex functions as a nucleating factor that organizes a subset of Ku70/80and PARP-1molecules into a DNA damage sensory complex in-volved in the surveillance of genome integrity.

Our fractionation studies indicate that WRN co-elutes with subpopulations of Ku70/80and PARP-1.It is important to note that coincidence of the peaks of each factor in the gel-filtration chromatography is not expected,as this would suggest that the majority of all three proteins exist in a complex.Such a result is not anticipated,as it is known that all three proteins interact with different partners in response to different physiological cues(24,53,54).Importantly,to verify that complex formation was not an artifact of the three factors independently binding to DNA,immunoprecipitation experiments were carried out in the presence of DNase I.These experiments yielded identical results(data not shown).The immunoprecipitation of WRN from eluates of fractionated extracts show that WRN forms two complexes with Ku,one of which contains PARP-1.These find-ings may indicate that there are two distinct WRN-Ku com-plexes in the cell.Alternatively,the association of PARP-1with Ku70/80and WRN may be part of a dynamic process that involves the rapid assembly and disassembly of this complex in the cell in response to diverse physiological and pathological stimuli.Clearly,we cannot rule out that the two observed WRN-Ku complexes may only be a consequence of the fraction-ation experiments;future studies will address this issue. PARP-1has been implicated in the protection of genome integrity by facilitating the repair of damaged DNA.In addi-tion,PARP-1participates in the execution of the apoptotic and necrotic pathways in cells with excessive DNA damage(55–57). The necrotic function of PARP-1has been linked to overactiva-tion of its enzymatic activity,which leads to the cellular deple-tion of NAD?and ATP(32,54,55).Excessive PARP-1activity has been implicated in the pathogenesis of several clinical conditions such as stroke,myocardial infarction,arthritis,di-abetes,and neurodegenerative disorders(54).The molecular mechanisms that regulate PARP-1survival and death-promot-ing effects are poorly defined;thus,it is possible that WRN and Ku70/80may play a role in the selection of the specific pathway that is activated in response to DNA damage.

PARP-1and poly(ADP-ribosyl)ation have been linked to mammalian longevity,as differences in the catalytic activity of PARP-1correlate with differences in life span between short and long-lived species(58).Because WS is a premature aging disease,and inactivation of Ku in mice leads to premature aging(27,28,31),the identification of a physical interaction between WRN,Ku70/80,and PARP-1suggests that these pro-teins are caretakers that function together in a cellular path-way that monitors the integrity of the genome and the longev-ity potential of an organism.Future biochemical and genetic studies will help in elucidating the link between the WRN complex and the network of cellular pathways that control the fate of cells with damaged DNA and shortened life spans.

Acknowledgments—We thank J.Mullenders for the preparation of the antigen used in the production of WRN antibody,C.Mazurek for help in the production of recombinant lentiviruses,and S.Reddy and members of the Comai lab for helpful discussions.

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日常工作中常用函数

AVERAGE函数主要功能：求出所有参数的算术平均值。使用格式：AVERAGE(number1,number2,……) 参数说明：number1,number2,……：需要求平均值的数值或引用单元格（区域），参数不超过30个。应用举例：在B8单元格中输入公式：=AVERAGE(B7：D7,F7：H7,7,8)，确认后，即可求出B7至D7区域、F7至H7区域中的数值和7、8的平均值。特别提醒：如果引用区域中包含“0”值单元格，则计算在内；如果引用区域中包含空白或字符单元格，则不计算在内。 COUNTIF函数主要功能：统计某个单元格区域中符合指定条件的单元格数目。使用格式：COUNTIF(Range,Criteria) 参数说明：Range代表要统计的单元格区域；Criteria表示指定的条件表达式。应用举例：在C17单元格中输入公式：=COUNTIF(B1：B13,">=80")，确认后，即可统计出B1至B13单元格区域中，数值大于等于80的单元格数目。特别提醒：允许引用的单元格区域中有空白单元格出现 DATEDIF函数主要功能：计算返回两个日期参数的差值。使用格式：=DATEDIF(date1,date2,"y")、=DATEDIF(date1,date2,"m")、=DATEDIF(date1,date2,"d") 参数说明：date1代表前面一个日期，date2代表后面一个日期；y（m、d）要求返回两个日期相差的年（月、天）数。

应用举例：在C23单元格中输入公式：=DATEDIF(A23,TODAY(),"y")，确认后返回系统当前日期[用TODAY()表示）与A23单元格中日期的差值，并返回相差的年数。特别提醒：这是Excel中的一个隐藏函数，在函数向导中是找不到的，可以直接输入使用，对于计算年龄、工龄等非常有效。 IF函数主要功能：根据对指定条件的逻辑判断的真假结果，返回相对应的内容。使用格式：=IF(Logical,Value_if_true,Value_if_false) 参数说明：Logical代表逻辑判断表达式；Value_if_true表示当判断条件为逻辑“真（TRUE）”时的显示内容，如果忽略返回“TRUE”；Value_if_false表示当判断条件为逻辑“假（FALSE）”时的显示内容，如果忽略返回“FALSE”。应用举例：在C29单元格中输入公式：=IF(C26>=18,"符合要求","不符合要求")，确信以后，如果C26单元格中的数值大于或等于18，则C29单元格显示“符合要求”字样，反之显示“不符合要求”字样。特别提醒：本文中类似“在C29单元格中输入公式”中指定的单元格，读者在使用时，并不需要受其约束，此处只是配合本文所附的实例需要而给出的相应单元格，具体请大家参考所附的实例文件。 INDEX函数主要功能：返回列表或数组中的元素值，此元素由行序号和列序号的索引值进行确定。使用格式：INDEX(array,row_num,column_num) 参数说明：Array代表单元格区域或数组常量；Row_num表示指定的行序号

to_char函数

表5-7. 格式化函数函数返回描述例子 to_char(timestamp, text) text 把timestamp 转换成string to_char(timestamp 'now','HH12:MI:SS') to_char(int, text) text 把int4/int8 转换成string to_char(125, '999') to_char(float, text) text 把float4/float8 转换成string to_char(125.8, '999D9') to_char(numeric, text) text 把numeric 转换成string to_char(numeric '-125.8', '999D99S') to_date(text, text) date 把string 转换成 date to_date('05 Dec 2000', 'DD Mon YYYY') to_timestamp(text, text) date 把string 转换成 timestamp to_timestamp('05 Dec 2000', 'DD Mon YYYY') to_number(text, text) numeric 把string 转换成 numeric to_number('12,454.8-', '99G999D9S') 表5-8. 用于date/time 转换的模板模板描述 HH 一天的小时数(01-12) HH12 一天的小时数(01-12) HH24 一天的小时数(00-23) MI 分钟(00-59) SS 秒(00-59) SSSS 午夜后的秒(0-86399) AM or A.M. or PM or P.M. 正午标识（大写） am or a.m. or pm or p.m. 正午标识（小写） Y,YYY 带逗号的年（4 和更多位）YYYY 年（4和更多位） YYY 年的后三位 YY 年的后两位 Y 年的最后一位 BC or B.C. or AD or A.D. 年标识（大写） bc or b.c. or ad or a.d. 年标识（小写）

Excel中常用函数及其使用方法简介

目录一、IF函数——————————————————————————————————2 二、ASC函数—————————————————————————————————4 三、SEARCH函数——————————————————————————————4 四、CONCATENATE函数———————————————————————————4 五、EXACT函数———————————————————————————————5 六、find函数—————————————————————————————————5 七、PROPER函数——————————————————————————————7 八、LEFT函数————————————————————————————————7 九、LOWER函数———————————————————————————————7 十、MID函数————————————————————————————————8 十一、REPT函数———————————————————————————————8 十二、Replace函数——————————————————————————————9 十三、Right函数———————————————————————————————10 十四、UPPER函数——————————————————————————————10 十五、SUBSTITUTE函数———————————————————————————10 十六、VALUE函数——————————————————————————————12 十七、WIDECHAR函数———————————————————————————12 十八、AND函数———————————————————————————————12 十九、NOT函数———————————————————————————————13 二十、OR函数————————————————————————————————13 二十一、COUNT函数—————————————————————————————14 二十二、MAX函数——————————————————————————————15 二十三、MIN函数——————————————————————————————15 二十四、SUMIF函数—————————————————————————————16 二十五、OFFSET函数————————————————————————————17 二十六、ROW函数——————————————————————————————20 二十七、INDEX 函数————————————————————————————21 二十八、LARGE函数—————————————————————————————22 二十九、ADDRESS函数————————————————————————————23 三十、Choose函数——————————————————————————————24 三十一、HLOOKUP函数———————————————————————————24 三十二、VLOOKUP函数———————————————————————————26 三十三、LOOKUP函数————————————————————————————29 三十四、MATCH函数————————————————————————————29 三十五、HYPERLINK函数——————————————————————————30 三十六、ROUND函数————————————————————————————31 三十七、TREND函数—————————————————————————————32

共同但有区别责任原则

题目：共同但有区别的责任原则在实施中的困境与对策姓名：罗珠玉、戴政

共同但有区别的责任原则在实施中的困境与对策摘要：共同但有区别的责任原则作为国际环境法的一项基本原则，该原则的要求在实践中未能得到充分尊重与落实。笔者通过对该原则实施困境及原因的分析,寻求解决该原则的可行性办法。关键词：共同但有区别的责任原则；实施困境；可行性办法

共同责任和区别责任组成了共同但有区别责任原则。二者之间相辅相成，密不可分。一方面各国不能以任何的借口而拒绝参与环境保护问题，这是每个国家的共同责任；另一方面，基于合理性而产生的区别责任，我们在对待共同责任的同时要给予发达国家与发展中国家差别待遇。只有当我们正确的理解二者关系时才能确保该原则的正确实施。实践中，该原则面对来自不同国家的阻力。一、共同但有区别的责任原则的实施困境发达国家有先进的技术与雄厚的资金，在各国订立国际公约之初，对发达国家明确规定了需向发展中国家提供环保技术的援助。可公约本身并未说明具体的援助方式，使发达国家有机可趁，利用市场操作以高价的方式向发展中国家提供商业性援助。而即使存在无偿性援助，实际数据也令人心寒，发展中国家适应气候变化每年所需的资金大约在 500 亿美元，而联合国的专门基金从发达国家筹集到的资金从 90 年代初至今总计只有 670 亿美元，发达国家对发展中国家的资金援助可见一斑，这也是共同但有区别责任难以落实的一个重要原因。在发展中国家共同但有区别的责任原则的实施也受到了挑战。发展中国家的经济水平比较落后，他们没有先进的技术支撑他们在保证解决自己温饱问题的同时兼顾环境保护，而要想解决生存问题必须以牺牲坏境为代价。传统的经济发展技术、能源技术已经不能适应现代可持续发展的要求，尤其 21 世纪对各国高新技术提出了更高的要求，在环境治理方面也不例外。现在单纯的现有技术转让已经不能满足发展中国家环境治理的需要了，发达国家需要尽可能地多与发展中国家进行技术交流与合作，让发展中国家也成为高新技术开发的参与者，掌握自主的知识产权。最后，为应对国际环境的问题而制定的众多国际公约，足以应对坚持和实施共同但有区别的责任原则。比如《人类环境宣言》、《联合国气候变化框架公约》、《联合国海洋法公约》、《京都议定书》······这些制定与签署的国际公约，不仅构成了世界环境保护国家合作的标准，而且也未共同但有区别责任作出了各种细化的规定。二、共同但有区别的责任原则实施中存在困难的原因美国曾以不符合本国的国家利益为由退出《京都议定书》，而各国对其只能进行谴责，因为国家享有主权原则，有权决定自己是否愿意加入某一国际公约。

Excel常用函数及使用方法

excel常用函数及使用方法一、数字处理（一）取绝对值：=ABS(数字) （二）数字取整：=INT(数字) （三）数字四舍五入：=ROUND(数字,小数位数) 二、判断公式（一）把公式返回的错误值显示为空： 1、公式：C2=IFERROR(A2/B2,"") 2、说明：如果是错误值则显示为空，否则正常显示。（二）IF的多条件判断 1、公式：C2=IF(AND(A2<500,B2="未到期"),"补款","") 2、说明：两个条件同时成立用AND,任一个成立用OR函数。三、统计公式（一）统计两表重复 1、公式:B2=COUNTIF(Sheet15!A:A,A2) 2、说明：如果返回值大于0说明在另一个表中存在，0则不存在。（二）统计年龄在30~40之间的员工个数公式=FREQUENCY(D2:D8,{40,29} （三）统计不重复的总人数 1、公式：C2=SUMPRODUCT(1/COUNTIF(A2:A8,A2:A8)) 2、说明：用COUNTIF统计出每人的出现次数，用1除的方式把出现次数变成分母，然后相加。

（四）按多条件统计平均值 =AVERAGEIFS(D:D,B:B,"财务",C:C,"大专") （五）中国式排名公式 =SUMPRODUCT(($D$4:$D$9>=D4)*(1/COUNTIF(D$4:D$9,D$4:D$9))) 四、求和公式（一）隔列求和 1、公式：H3=SUMIF($A$2:$G$2,H$2,A3:G3) 或=SUMPRODUCT((MOD(COLUMN(B3:G3),2)=0)*B3:G3) 2、说明：如果标题行没有规则用第2个公式（二）单条件求和 1、公式：F2=SUMIF(A:A,E2,C:C) 2、说明：SUMIF函数的基本用法（三）单条件模糊求和说明：如果需要进行模糊求和，就需要掌握通配符的使用，其中星号是表示任意多个字符，如"*A*"就表示a前和后有任意多个字符，即包含A。（四）多条求模糊求和 1、公式：=SUMIFS(C2:C7,A2:A7,A11&"*",B2:B7,B11) 2、说明：在sumifs中可以使用通配符* （五）多表相同位置求和 1、公式：=SUM(Sheet1:Sheet19!B2) 2、说明：在表中间删除或添加表后，公式结果会自动更新。

论共同但有区别责任原则在我国的适用(改)

论共同但有区别责任原则在我国的适用摘要: 文章在结合我国的具体国情的基础上,对我国进行环境污染防治过程中在环境保护法律中适用这一前沿的原则所具有的理论基础以及需要注意的问题进行了探讨。关键词:京都议定书;共同但有区别责任原则;共同责任;区别责任 1 共同但有区别责任原则概述共同但有区别责任原则是国际环境法中的一项基本原则。这一原则的产生主要是基于各国社会发展的历史对国际环境的影响及本国的实际承担能力。其核心思想是,在实现将大气中温室气体的浓度稳定在防止气候系统受到危险的人为干扰的水平上这一目标过程中全球各国都负有共同的责任和义务,但是基于各国的历史发展状况及现实承受能力,发达国家应该在这一过程中应该率先承担并且承担主要的责任。 1.1共同但有区别的责任原则主要包含以下两个基本要素： 1.1.1共同责任共同责任的理论依据:全球的生态系统是一个不可分割的整体,环境问题具有全球性,解决全球环境问题需要所有国家的参与，每个国家都有责任。全球环保问题已经成为人类共同关注的焦点,而不只是某一个国家的国内立法问题。共同责任的内容:许多关于环境与发展的国际文件中均有共同责任的规定。共同责任要求每个国家不论其大小、贫富等方面的区别,都对保护全球环境负有一份责任,都应当参加全球环境保护事业,都必须在保护和改善环境方面承担义务。基于共同责任,所有国家,尤其是发展中国家,都应该参与关于可持续发展的立法以及相关法律的实施。许多现有的有关环境的国际法律文件没有发展中国家的参与。为了保护发展中国家的利益,有必要对相关文件进行修订，从而确保上述法律文件适用范围的广泛性。 1.1.2有区别的责任有区别的责任的理论依据:有区别的责任的理论依据是公平原则。如果一个国家曾未经其他国家同意而不公平地对其进行利用而使其付出代价,那么受害国有权要

C语言常用函数手册

1.分类函数,所在函数库为ctype.h int isalpha(int ch) 若ch是字母('A'-'Z','a'-'z')返回非0值,否则返回0 int isalnum(int ch) 若ch是字母('A'-'Z','a'-'z')或数字('0'-'9'),返回非0值,否则返回0 int isascii(int ch) 若ch是字符(ASCII码中的0-127)返回非0值,否则返回0 int iscntrl(int ch) 若ch是作废字符(0x7F)或普通控制字符(0x00-0x1F) 返回非0值,否则返回0 int isdigit(int ch) 若ch是数字('0'-'9')返回非0值,否则返回0 int isgraph(int ch) 若ch是可打印字符(不含空格)(0x21-0x7E)返回非0值,否则返回0 int islower(int ch) 若ch是小写字母('a'-'z')返回非0值,否则返回0 int isprint(int ch) 若ch是可打印字符(含空格)(0x20-0x7E)返回非0值,否则返回0 int ispunct(int ch) 若ch是标点字符(0x00-0x1F)返回非0值,否则返回0 int isspace(int ch) 若ch是空格(' '),水平制表符('\t'),回车符('\r'), 走纸换行('\f'),垂直制表符('\v'),换行符('\n') 返回非0值,否则返回0 int isupper(int ch) 若ch是大写字母('A'-'Z')返回非0值,否则返回0 int isxdigit(int ch) 若ch是16进制数('0'-'9','A'-'F','a'-'f')返回非0值, 否则返回0 int tolower(int ch) 若ch是大写字母('A'-'Z')返回相应的小写字母('a'-'z') int toupper(int ch) 若ch是小写字母('a'-'z')返回相应的大写字母('A'-'Z') 2.数学函数,所在函数库为math.h、stdlib.h、string.h、float.h int abs(int i) 返回整型参数i的绝对值 double cabs(struct complex znum) 返回复数znum的绝对值 double fabs(double x) 返回双精度参数x的绝对值 long labs(long n) 返回长整型参数n的绝对值 double exp(double x) 返回指数函数ex的值 double frexp(double value,int *eptr) 返回value=x*2n中x的值,n存贮在eptr中double ldexp(double value,int exp); 返回value*2exp的值 double log(double x) 返回logex的值 double log10(double x) 返回log10x的值 double pow(double x,double y) 返回xy的值 double pow10(int p) 返回10p的值 double sqrt(double x) 返回+√x的值 double acos(double x) 返回x的反余弦cos-1(x)值,x为弧度 double asin(double x) 返回x的反正弦sin-1(x)值,x为弧度 double atan(double x) 返回x的反正切tan-1(x)值,x为弧度 double atan2(double y,double x) 返回y/x的反正切tan-1(x)值,y的x为弧度double cos(double x) 返回x的余弦cos(x)值,x为弧度 double sin(double x) 返回x的正弦sin(x)值,x为弧度 double tan(double x) 返回x的正切tan(x)值,x为弧度 double cosh(double x) 返回x的双曲余弦cosh(x)值,x为弧度 double sinh(double x) 返回x的双曲正弦sinh(x)值,x为弧度

Excel常用函数的使用方法

1、ABS函数函数名称：ABS 主要功能：求出相应数字的绝对值。使用格式：ABS(number) 参数说明：number代表需要求绝对值的数值或引用的单元格。应用举例：如果在B2单元格中输入公式：=ABS(A2)，则在A2单元格中无论输入正数（如100）还是负数（如-100），B2中均显示出正数（如100）。特别提醒：如果number参数不是数值，而是一些字符（如A等），则B2中返回错误值“#VALUE！”。 2、AND函数函数名称：AND 主要功能：返回逻辑值：如果所有参数值均为逻辑“真（TRUE）”，则返回逻辑“真（TRUE）”，反之返回逻辑“假（FALSE）”。使用格式：AND(logical1,logical2, ...) 参数说明：Logical1,Logical2,Logical3……：表示待测试的条件值或表达式，最多这30个。应用举例：在C5单元格输入公式：=AND(A5>=60,B5>=60)，确认。如果C5中返回TRUE，说明A5和B5中的数值均大于等于60，如果返回FALSE，说明A5和B5中的数值至少有一个小于60。特别提醒：如果指定的逻辑条件参数中包含非逻辑值时，则函数返回错误值“#VALUE!”或“#NAME”。 3、AVERAGE函数函数名称：AVERAGE 主要功能：求出所有参数的算术平均值。使用格式：AVERAGE(number1,number2,……) 参数说明：number1,number2,……：需要求平均值的数值或引用单元格（区域），参数不超过30个。

应用举例：在B8单元格中输入公式：=AVERAGE(B7:D7,F7:H7,7,8)，确认后，即可求出B7至D7区域、F7至H7区域中的数值和7、8的平均值。特别提醒：如果引用区域中包含“0”值单元格，则计算在内；如果引用区域中包含空白或字符单元格，则不计算在内。 4、COLUMN 函数函数名称：COLUMN 主要功能：显示所引用单元格的列标号值。使用格式：COLUMN(reference) 参数说明：reference为引用的单元格。应用举例：在C11单元格中输入公式：=COLUMN(B11)，确认后显示为2（即B列）。特别提醒：如果在B11单元格中输入公式：=COLUMN()，也显示出2；与之相对应的还有一个返回行标号值的函数——ROW(reference)。 5、CONCATENATE函数函数名称：CONCATENATE 主要功能：将多个字符文本或单元格中的数据连接在一起，显示在一个单元格中。使用格式：CONCATENATE(Text1，Text……) 参数说明：Text1、Text2……为需要连接的字符文本或引用的单元格。应用举例：在C14单元格中输入公式：=CONCATENATE(A14,"@",B14,".com")，确认后，即可将A14单元格中字符、@、B14单元格中的字符和.com连接成一个整体，显示在C14单元格中。特别提醒：如果参数不是引用的单元格，且为文本格式的，请给参数加上英文状态下的双引号，如果将上述公式改为：=A14&"@"&B14&".com"，也能达到相同的目的。 6、COUNTIF函数函数名称：COUNTIF 主要功能：统计某个单元格区域中符合指定条件的单元格数目。使用格式：COUNTIF(Range,Criteria) 参数说明：Range代表要统计的单元格区域；Criteria表示指定的条件表达式。

EXCEL中常用函数及使用方法

EXCEL中常用函数及使用方法 Excel函数一共有11类：数据库函数、日期与时间函数、工程函数、财务函数、信息函数、逻辑函数、查询和引用函数、数学和三角函数、统计函数、文本函数以及用户自定义函数。 1.数据库函数当需要分析数据清单中的数值是否符合特定条件时，可以使用数据库工作表函数。例如，在一个包含销售信息的数据清单中，可以计算出所有销售数值大于1,000 且小于2,500 的行或记录的总数。Microsoft Excel 共有12 个工作表函数用于对存储在数据清单或数据库中的数据进行分析，这些函数的统一名称为Dfunctions，也称为D 函数，每个函数均有三个相同的参数：database、field 和criteria。这些参数指向数据库函数所使用的工作表区域。其中参数database 为工作表上包含数据清单的区域。参数field 为需要汇总的列的标志。参数criteria 为工作表上包含指定条件的区域。 2.日期与时间函数通过日期与时间函数，可以在公式中分析和处理日期值和时间值。 3.工程函数工程工作表函数用于工程分析。这类函数中的大多数可分为三种类型：对复数进行处理的函数、在不同的数字系统（如十进制系统、十六进制系统、八进制系统和二进制系统）间进行数值转换的函数、在不同的度量系统中进行数值转换的函数。 4.财务函数财务函数可以进行一般的财务计算，如确定贷款的支付额、投资的未来值或净现值，以及债券或息票的价值。财务函数中常见的参数：未来值(fv)--在所有付款发生后的投资或贷款的价值。期间数(nper)--投资的总支付期间数。付款(pmt)--对于一项投资或贷款的定期支付数额。现值(pv)--在投资期初的投资或贷款的价值。例如，贷款的现值为所借入的本金数额。利率(rate)--投资或贷款的利率或贴现率。类型(type)--付款期间内进行支付的间隔，如在月初或月末。 5.信息函数可以使用信息工作表函数确定存储在单元格中的数据的类型。信息函数包含一组称为IS 的工作表函数，在单元格满足条件时返回TRUE。例如，如果单元格包含一个偶数值，ISEVEN 工作表函数返回TRUE。如果需要确定某个单元格区域中是否存在空白单元格，可以使用COUNTBLANK 工作表函数对单元格区域中的空白单元格进行计数，或者使用ISBLANK 工作表函数确定区域中的某个单元格是否为空。 6.逻辑函数使用逻辑函数可以进行真假值判断，或者进行复合检验。例如，可以使用IF 函数确定条件为真还是假，并由此返回不同的数值。

论共同但有区别责任原则

论共同但有区别责任原则 ——全球环境徐博（机械与汽车工程系机制2082班）摘要:文章在结合我国的具体国情的基础上,对我国进行环境污染防治过程中在环境保护法律中适用这一前沿的原则所具有的理论基础以及需要注意的问题进行了探讨。关键词:京都议定书;共同但有区别责任原则一、共同但有区别责任原则的主要内容《京都议定书》第一次设定了具有法律约束力的温气限排额度,是迄今为止国际社会承诺削减温气排放、遏制地球变暖的唯一一项国际公约。结合1994年3月生效的《联合国气候变化框架国际公约》的相关内容可知,共同但有区别责任原则主要内容包含两个方面——共同责任以及有区别责任。由于现实原因的限制或者说是从公平的角度考虑,发达国家和发展中国家在国际环境保护中所要承担的责任的范围、时间、方式、手段等方面是有差异的,从历史和现实的角度出发,对于各国的具体责任的确定,应当兼顾公平与效率,统筹考虑各种因素,在公平和效率之间做出适当的权衡取舍。保护和改善全球环境是全人类的共同利益所在,是世界各国的共同责任。这种共同责任主要体现在:基于“地区生态系统的整体性”,各国,不论其大小、贫富方面的差别都应该采取措施保护和改善其管辖范围内的环境,并防止对管辖范围以外的环境造成损害,同时各国应该在环境方面相互合作和支持等。但是另一方面,由于各国经济发展和工业化的水平不同,废弃物和污染物的排放数量也不同,不应该要求所有的国家承担完全相同的责任。发达国家在自身发展过程中曾经向大气排放大量有害物质,最先并且主要是他们造成了大气的污染,发展中国家不应为他们造成的大气污染后果承担责任。二、共同但有区别责任原则适用于我国环境法律体系的基础不可否认,共同但有区别责任原则在全球范围内是适用、且必须加以运用的。一种被证实具有优越性的原则能否在我国的环境法中适用,必须要针对我国的具体国情以及此原则的特征进行分析。 (一)我国在环境保护方面与世界进行了深入的交流和合作,具备运用相应知识的能力从环境角度来看,世界是一体的,一国环境的污染和破坏都可能引起相关地区甚至全球范围内的环境破坏。我国积极参加全球范围内的环境保护活动,签订相关的环境保护国际协议。我国先后与30多个国家签署了双边环境合作协议或备忘录,与美国、日本、法国、德国、加拿大、俄罗斯等10个国家签订了有关核安全与辐射环境管理的双边合作协议,与联合国环境规划署、联合国开发计划署、国际原子能机构、世界银行、亚洲开发银行、全球环境基金、蒙特利尔议定书多边基金等国际机构建立了密切的合作关系。积极参与了重要国际环境公约的谈判和重要多边环境论坛的活动,参加或签署了气候变化框架公约、生物多样性公约、保护臭氧层的维也纳公约和蒙特利尔议定书、巴塞尔公约、核安全公约等国际环境公约,广泛、深入地开展了有关国际公约的履约工作。表明我国在环境保护方面已经全面与世界接轨,对国际环境保护及其责任履行上的原则有了深入地了解和学习,能够结合我国的具体实际情况合理地移植到我国的相关法律体系中来。

WPS表格常用函数应用教程(经典版)

WPS表格常用函数应用教程一、函数应用基础（一）函数和公式 1.什么是函数 WPS表格函数即是预先定义，执行计算、分析等处理数据任务的特殊公式。以常用的求和函数SUM为例，它的语法是“SUM(数值1, 数值2,......)”。其中“SUM”称为函数名称，一个函数只有唯一的一个名称，它决定了函数的功能和用途。函数名称后紧跟左括号，接着是用逗号分隔的称为参数的内容，最后用一个右括号表示函数结束。参数是函数中最复杂的组成部分，它规定了函数的运算对象、顺序或结构等。使得用户可以对某个单元格或区域进行处理，如确定成绩名次、计算三角函数值等。 2.什么是公式函数与公式既有区别又互相联系。如果说前者是WPS 表格预先定义好的特殊公式，后者就是由用户自行设计对工作表进行计算和处理的公式。以公式“=SUM(E1:H1)*A1+26”为例，它要以等号“=”开始，其内部可以包括函数、引用、运算符和常量。上式中的“SUM(E1:H1)”是函数，“A1”则是对单元格A1 的引用(使用其中存储的数据)，“26”则是常量，“*”和

“+”则是算术运算符(另外还有比较运算符、文本运算符和引用运算符)。如果函数要以公式的形式出现，它必须有两个组成部分，一个是函数名称前面的等号，另一个则是函数本身。（二）函数的参数函数右边括号中的部分称为参数，假如一个函数可以使用多个参数，那么参数与参数之间使用半角逗号进行分隔。参数可以是常量(数字和文本)、逻辑值(例如真值或假值)、数组、错误值(例如#N/A)或单元格引用(例如E1:H1)，甚至可以是另一个或几个函数等。参数的类型和位置必须满足函数语法的要求，否则将返回错误信息。 1.常量常量是直接输入到单元格或公式中的数字或文本，或由名称所代表的数字或文本值，例如数字“2890.56”、日期“2003-8-19”和文本“黎明”都是常量。但是公式或由公式计算出的结果都不是常量，因为只要公式的参数发生了变化，它自身或计算出来的结果就会发生变化。 2.逻辑值逻辑值是比较特殊的一类参数，它只有真或假两种类型。例如在公式“=IF(A3=0,"",A2/A3)”中，“A3=0”就是一个可以返回真或假两种结果的参数。当“A3=0”为真时在公式所在单元格中填入“0”，否则在单元格中填入“A2/A3”的计算结果。

论国际环境法的共同但有区别责任原则

目录毕业论文诚信承诺书 (2) 摘要 (3) 关键字 (3) 正文一、共同但有区别责任原则的概述 (3) 二、共同但有区别责任原则的发展 (4) 三、共同但有区别责任原则的性质 (5) 四、共同但有区别责任原则的意义 (6) 五、坚持和发展“共同但有区别责任”原则 (6) 结语 (7) 参考文献 (7)

毕业论文诚信承诺书本人作为《论国际环境法的共同但有区别责任》一文的作者，郑重承诺：一、本论文是我在导师的指导下，参考相关文献资料，进行分析研究，独立完成的，其中所引用的文献资料和相关数据，都是真实的，除标明出处的内容外，不包含他人已公开发表的研究成果和学术观点。二、本论文中若有抄袭他人研究成果和剽窃他人学术观点，本人自愿承担取消毕业论文成绩、交回学历学位证书等一切后果。学生签名：年月日注：本承诺书一式二份，一份置于毕业论文分册首页，一份置于过程材料分册末页。

论国际环境法的共同但有区别责任原则摘要环境保护已经成为我们时代最为重大的主题之一。世界每一个成员都应当共同承担保护和改善全球环境的责任，环境保护不再是仅限于一个两个国家主权之间的事情。全球性环境问题需要所有国家的共同努力才能得以解决。在对环境问题形成所起的作用上，发达国家和发展中国家扮演者主次不同的角色。如果要让本来就相对贫困的发展中国家在解决目前的全球性问题上承担和发达国家同样的义务，肯定是不公平的，必然会遭到发展中国家的反对。国际环境法的共同但有区别的责任原则“就在这样的背景下产生了，它调解了国家之间的矛盾，促进各国都参与到全球环保事业当中，将不同国情，制度的国家团结成一个“求大同、存小异”合作的整体。共同担有区别的责任原则主要包含两层意思：共同责任和区别责任。它不但是国际法上的一项重要原则，更代表了环境争议思想在适用范围上的扩展，本文主要探讨共同但是有区别的责任的定义、内涵以及环境正义与共同但有区别的责任之间的关系，还有共同但有区别的责任定义的必要性。关键字共同但有区别的责任国际发达国家发展中国家一、共同但有区别责任原则的概述国际环境法中共同但有区别责任原则因体现谋求优先发展经济的利益诉求,获得大部分发展中国家认同。但该原则的地位、内容一直存有争议。随着中国、印度等发展中大国碳排放日益增长,在后续气候谈判中如仅以发展中国家身份不参加实质减排,将面临极大压力。因此,探讨该原则的地位以及承担责任的依据,对于确定发达国家与发展中国家应对气候变化所应承担的共同责任以及各自应承担的义务,将具有重要意义. 共同但有区别责任主要体现在国际气候大会中表现最为明显，备受关注的联合国第十九次气候变化大会雨2013年11月23日晚在波兰首都华沙落幕，会期比原计划拖延了一整天。经过长达两周的艰难谈判和激烈争吵，特别是会议结束前最后48小时，各国代表挑灯夜战，最终就德班平台决议、气候资金和损失损害补偿机制等焦点议题签署了协议。但是，由于发达国家不愿承担历史责任，在落实向发展中国家提供资金援助问题上没有诚信，导致政治互信缺失，加上个别发达国家的减排立场严重倒退，致使谈判数次陷入僵局。会议最终经过妥协，达成了各方都不满意、但都能够接受的结果。《联合国气候变化框架公约》第十九次缔约方会议暨《京都议定书》第九次缔约方会议23日晚打破僵局达成协议后在华沙落下帷幕。尽管大会成果不尽如人意，但中方表示，节能减排是中国可持续发展的内在要求，无论谈判进展如何

Oracleto_char格式化函数

表5-8. 用于date/time 转换的模板

所有模板都都允许使用前缀和后缀修改器。模板里总是允许使用修改器。前缀'FX' 只是一个全局修改器。

表5-9. 用于日期/时间模板to_char() 的后缀用法须知： ?如果没有使用FX选项，to_timestamp和to_date忽略空白。FX必须做为模板里的第一个条目声明。 ?反斜杠（"\"）必须用做双反斜杠（"\\"），例如'\\HH\\MI\\SS'。 ?双引号（'"'）之间的字串被忽略并且不被分析。如果你想向输出写双引号，你必须在双引号前面放置一个双反斜杠（'\\'），例如 '\\"YYYY Month\\"'。 ?to_char支持不带前导双引号（'"'）的文本，但是在双引号之间的任何字串会被迅速处理并且还保证不会被当作模板关键字解释（例如：'"Hello Year: "YYYY'）。表5-10. 用于to_char(numeric) 的模板

用法须知： ?使用'SG'，'PL' 或'MI' 的带符号字并不附着在数字上面；例如，to_char(-12, 'S9999') 生成' -12'，而to_char(-12, 'MI9999') 生成'- 12'。Oracle里的实现不允许在9前面使用MI，而是要求9在MI前面。 ?PL，SG，和TH是 Postgres 扩展。 ?9表明一个与在9字串里面的一样的数字位数。如果没有可用的数字，那么使用一个空白（空格）。 ?TH不转换小于零的值，也不转换小数。TH是一个 Postgres 扩展。 ?V方便地把输入值乘以10^n，这里n是跟在V后面的数字。to_char不支持把V与一个小数点绑在一起使用（例如. "99.9V99" 是不允许的）。表5-11. to_char例子

“共同但有区别的责任”原则的解读

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scanf()getchar()和gets()等函数的注意事项

------------------------------------------------------------------- | 问题描述一：（分析scanf()和getchar()读取字符）| ------------------------------------------------------------------- scanf(), getchar()等都是标准输入函数，一般人都会觉得这几个函数非常简单，没什么特殊的。但是有时候却就是因为使用这些函数除了问题，却找不出其中的原因。下面先看一个很简单的程序：程序1： #include int main() { char ch1, ch2; scanf("%c", &ch1); scanf("%c", &ch2); printf("%d %d\n", ch1, ch2); return 0; } 或者是： #include int main() { char ch1, ch2; ch1 = getchar();

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=COUNTIF(A1:A29,">=80")-COUNTIF(A1:A29," =90") 方法二：（1）=COUNTIF(A2:A56,"100") ——求A2到A56区域100分的人数；假设把结果存放于A57单元格；（2）=COUNTIF(A2:A56,">=95")－A57 ——求A2到A56区域大于等于95而小于100分的人数；假设把结果存放于A58单元格；（3）=COUNTIF(A2:A56,">=90")－SUM(A57:A58) ——求A2到A56区域大于等于90而小于95分的人数；假设把结果存放于A59单元格；（4）=COUNTIF(A2:A56,">=85")－SUM(A57:A59) ——求A2到A56区域大于等于85而小于90分的人数； …… 8、求A2到A56区域优秀率：=(COUNTIF(A2:A56,">=90"))/55*100 9、求A2到A56区域及格率：=(COUNTIF(A2:A56,">=60"))/55*100 10、排名公式： =RANK(A2，A$2:A$56) ——对55名学生的成绩进行排名； 11、标准差：=STDEV(A2:A56) ——求A2到A56区域(55人)的成绩波动情况（数值越小，说明该班学生间的成绩差异较小，反之，说明该班存在两极分化）； 12、条件求和：=SUMIF(B2:B56,"男"，K2:K56) ——假设B列存放学生的性别，K列存放学生的分数，则此函数返回的结果表示求该班男生的成绩之和；