Short communication
Quantitative analysis of WRN exonuclease activity by isotope dilution mass spectrometry
Aswin Mangerich
a,1,*, Sebastian Veith
a,1, Oliver Popp
a, Jo¨rg Fahrer
a,2, Rita Martello
a, Vilhelm A. Bohr
b, Alexander Bu¨rkle
a,*
aMolecularToxicologyGroup,DepartmentofBiology,UniversityofKonstanz,78457Konstanz,Germany
bLaboratoryofMolecularGerontology,BiomedicalResearchCenter,NationalInstituteonAging,NIH,Baltimore,MD21224,USA
Wernersyndrome(WS)isarareautosomalrecessivedisorder characterized by a segmental premature aging phenotype, includingearlyonsetofatherosclerosis,osteoporosis,andahigh cancer incidence. The disease is caused by loss-of-function mutationsin theWRNgenewhich encodestheWRNprotein,a member of the RecQ helicase family. On the cellular level, fibroblastsderivedfromWSpatientsdisplaygenomicinstability and a reduced replicative life span (Kudlow et al., 2007). This phenotypeisinaccordancewithexperimentaldatademonstrating thatWRNisinvolvedinmultipleaspectsofDNAmetabolism,such asDNAreplication,genomicmaintenance,andtelomereregulation (Bohr,2008;Reddyetal.,2010;Rossietal.,2010).Incontrasttothe otherfivemembersofthehumanRecQhelicasefamily,WRNalso possesses a unique 30!50 exonuclease activity (Huang et al., 1998).
TheWRNexonucleasecleavestheDNAphosphodiesterbond andreleasesfree 50-dNMPsfromtheDNA strand(Kamath-Loeb etal.,1998).Toelicititsexonucleaseactivity,WRNrequiresa30 recessed end (50-overhang) substrate. WRN does not degrade duplexDNAwithbluntends,unlessthesubstratealsocontainsa junctionoralternateDNAstructuressuchasafork(Broshetal., 2006;ShenandLoeb,2000).Itislargelyinactiveonshortsingle- strandedDNA substrates(Kamath-Loebetal.,1998),but longer ssDNAsubstratesareefficientlydegraded(Machweetal.,2006).Its activityisregulatedbyposttranslationalmodificationsandprotein interactions. For instance, phosphorylation of WRNby DNA-PK inhibitsitsexonucleaseactivity(Karmakaretal.,2002;Yannone etal.,2001). Inaddition,p53,BLM,andPARP1causeinhibitory effects(Broshetal.,2001;Sommersetal.,2005;vonKobbeetal., 2002,2004),whereastheKu70/80complexstimulatesexonucle- aseactivity(Cooperetal.,2000;Kudlowetal.,2007;LiandComai, 2000,2001).
StandardmethodstoassessWRNexonucleaseactivityutilize radioactively or fluorescently 50 end-labeled DNA substrates to detectthedegradationofthefull-lengthDNAmolecules(Boubriak etal., 2009;Broshetal.,2006).Here wepresentan alternative approach toassess WRNexonuclease activitybased on isotope dilution mass spectrometry (LC–MS/MS). This method may be Keywords:
Wernersyndrome WRN
Exonuclease Massspectrometry Aging
PARP1
ABSTRACT
Wernersyndromeisadisordercharacterizedbyaprematureagingphenotype.Thediseaseiscausedby mutationsintheWRNgenewhichencodesaDNAhelicase/exonucleasewhichisinvolvedinmultiple aspectsofDNAmetabolism.CurrentmethodsmostlyrelyonradiometrictechniquestoassessWRN exonucleaseactivity.Herewepresentanalternative,quantitativeapproachbasedonnon-radioactive isotopedilutionmassspectrometry (LC–MS/MS).A oligoduplexsubstratemimicking thetelomeric sequencewasusedformethoddevelopment.Releasednucleotides,whichcorrelatewiththedegreeof oligoduplex degradation, were dephosphorylated, purified, and quantified by LC–MS/MS. Heavy- isotope-labeledinternalstandardswereused toaccountfortechnicalvariability. Themethodwas validatedintermsofreproducibility,time-courseandconcentration-dependencyofthereaction.As showninthisstudy,theLC–MS/MSmethodcanassessexonucleaseactivityofWRNmutants,WRN’s substrate and strand specificity, and modulatory effects of WRN interaction partners and posttranslationalmodifications.Moreover,itcanbeusedtoanalyzetheselectivityandprocessivity ofWRNexonucleaseand allowsthescreeningofsmallmoleculesforWRNexonucleaseinhibitors.
Importantly,thisapproachcaneasilybeadaptedtostudynucleasesotherthanWRN.Thisisofgeneral interest,becauseexonucleasesarekeyplayersinDNAmetabolismandagingmechanisms.
*Correspondingauthorsat:MolecularToxicologyGroup,DepartmentofBiology, UniversityofKonstanz,D-78457Konstanz,Germany.
E-mailaddresses:aswin.mangerich@uni-konstanz.de(A.Mangerich), alexander.buerkle@uni-konstanz.de(A.Bu¨rkle).
1Bothauthorscontributedequally.
2Presentaddress:InstituteofToxicology,UniversityMedicalCenterMainz,D- 55131Mainz,Germany.
Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-197064
Erschienen in: Mechanisms of Ageing and Development ; 133 (2012), 8. - S. 575-579 https://dx.doi.org/10.1016/j.mad.2012.06.005
particularlyusefulintwosituations:Firstly,forlaboratoriesthat wish to replace the common radioactive assays with a non- radioactiveoneand,secondly,themethodcanbeincorporatedinto high throughput screeningapproaches forsmallmolecules that affectexonucleaseactivity.
Wehavevalidatedournewlydevelopedmethodandcompared ittoamodifiedversionofanestablishedprotocolthatusesa50- biotin-end-labeledDNAsubstratetodetectactivityofrecombinant WRNexonuclease(Broshetal.,2006)(Suppl.Fig.1).Importantly, using a telomericsubstratemimics oneofthekey functionsof WRNwhichistooperateatthetelomere(Bohr,2008).Toassessif thisoligoduplexindeedservesasasuitablesubstrateforWRNin ourhands,anexonucleasereactionwascarriedoutaspublished previously (Broshet al., 2006). Thereaction mixturecontained 75fmoloftheoligoduplexand0.1–1pmolofrecombinantWRN.
Subsequently, digestion products were resolved by denaturing polyacrylamidegelelectrophoresis(PAGE)andbiotinwasdetected bystreptavidin-POD(Suppl.Fig.2).Inthismethod,lossofsignal intensityofthefull-lengthend-labeledDNAsubstratewasusedas readouttoassessexonucleaseactivity.AsisevidentfromSuppl.
Fig.2,WRNefficientlycatalyzesthedegradationofthisoligodu- plex to truncated DNA molecules of various lengths in a concentration dependent manner. Initial degradation of the substrate is visible atan enzymeto substrateratio (E/S)of 1 (10nMWRN)andreachedsaturationatanE/Sof8(60nMWRN) withamaximumefficiencyof80%.
InsteadofdetectingtheshortenedDNAsubstrate,therationale of the LC–MS/MS-based method is to detectdegradation end- products,i.e.,freenucleosides,toassessWRNexonucleaseactivity.
Toallowcomparabilitytothebiotin-end-labelingtechnique,the samesubstrateandidenticalreactionconditionswerechosento develop the LC–MS/MS-based method. Since the oligoduplex contains a repetitivetelomericsequence(TTAGGG)4,LC–MS/MS quantificationoffree20-deoxyguanosine(dG)wasexpectedtobea suitablereadouttoassessWRNexonucleaseactivity.Fig.1showsa flow chartoftheexperimentalprocedure;adetailedprotocolis availableinthesupplementaryinformationsection.Briefly,after theexonucleasereactionwascarriedout,sampleswereplacedon iceand15N-labeleddGwasaddedasaninternalstandardtothe
reactionmixturetoaccountfortechnicalvariabilityduringsample work-up and mass spectrometric measurement. Thereafter, recombinant WRN was removed by spin column filtration, followedbydephosphorylationofthenucleotidestonucleosides usingalkalinephosphatase(AP),removalofthephosphataseby spincolumn filtration, andsubsequentLC–MS/MSanalysis.The recoveryrateoftheinternalstandardwasusually70%.Typical LC–MS/MSchromatograms of unlabeled and 15N-labeleddG as wellasacalibrationcurveareshowninSuppl.Fig.3.
Themethodshowsadequateassay-to-assayvariability(Suppl.
Fig.4A)andcanbeperformedin1–2daysdependentonthetime chosen for AP digestion (Suppl. Fig. 4B; NB: no significant differencesin thequantitiesof dGwereobserved between4-h andovernightAPdigestion,indicatingthatanAPdigestiontimeof 4hissufficientforcompletedephosphorylationofdGMP).
As shown in Fig. 2, free dG was detected in a time and concentrationdependentmanner.Sincethereactionisstillinits dynamic range after 15min in terms of release of free dGMP (Fig.2A),areactiontimeof45minwaschosenforthefollowing experiments to achieve maximum oligoduplex degradation. In agreementwithresultsobtainedfrombiotin-end-labeling tech- nique,WRNactivitywasalreadydetectedatanE/Sof1(5–10nM WRN) and reached saturation at an E/S of 8 (60nM WRN) (Fig.2B).Amaximumof32%oftheexpectedtotalamountofdG wasdetected inWRN-digested samples.Thelowerefficiencyof WRN compared tophosphodiesterase (PDE) may berelated to incomplete annealing of the DNA strands and therefore to incompletedigestionbyWRN.A WRNmutant withamutation in theexonucleasedomain(WRN-E84A, X-WRN)(Huang etal., 1998;Machweetal.,2000)showedstronglydiminishedexonu- clease activity compared to WT-WRN (Fig. 2C). The residual exonuclease activity observed in the X-WRN sample may be related to the sensitivity of the LC–MS/MS method, to some contamination with Zn2+ ions, which can trigger minimal exonuclease activity of X-WRN (Choudhary et al., 2004), or to contaminationoftheX-WRNproteinwithanunknownnuclease.
Conceivable applications of the LC–MS/MS-based method includestudyingeffectsoffactorsthatmodulateWRNexonuclease activity, such as posttranslational modifications and protein interaction partners. For example, PARP1, which catalyzes the synthesis of the biopolymer poly(ADP-ribose) upon genotoxic stress,isanestablishedinteractionpartnerofWRN(Rossietal., 2010;Rouleauetal.,2010).Previously,itwasshownthatPARP1 inhibits both WRN’s helicase and exonuclease activities (von Kobbeetal.,2004).Inagreementwiththeseresults,PARP1ledto aninhibitionofWRNexonucleaseactivitybyupto80%asdetected byLC–MS/MSanalysis(Fig.2D).
Furthermore, the method can be applied to study WRN’s substrate and strandspecificity. To this end, we tested several newlydesignedWRNoligoduplexsubstrateswhicharecompatible withourassay,i.e.anoptimizedforkedoligoduplex,ablunt-ended oligoduplex,andoligoduplexescontaininga4-wayjunctionanda 50-overhang(Suppl.Table1forfullsequencedetails).Allsubstrates compriseda strandthat contains only dG, dA, dT (‘dG’strand) within a repetitive sequence element (XXXGGG)6 (X=A,T in alternating sequence to assure annealing specificity) and a complementarystrandthatonlycontainsdC,dA,dT(‘dC’strand).
Exceptfortheblunt-endedsubstrate,thedG-strandwasexpected tobeaccessibleforWRNexonucleaseactivity.Inagreementwith previous reports(Broshetal., 2006;Kamath-Loebetal., 1998), oligoduplexeswhichcontainedtheforkand4-wayjunctionserved asefficientsubstratesfor WRNexonuclease,whereas theblunt endedoligoduplexshowedalmostnoreleaseoffreedG(Fig.3A).
Moreover,significantWRNexonucleaseactivitywasdetectedwith the50overhangsubstrate,howevertoalesserextentthanwiththe forkand4-wayjunctionoligoduplexes.Asexpected,WRNwasnot
Exonuc lease re actio n to g enerate free nucl eoti des
Remov al of enzymes by filtrati on through 10 kD c ut-off filter Addition of
15N-labeled inter nal sta ndard
Dephos phorylation of nucl eoti des to nucl eos ides
Remov al of phos phatas e by filtrati on through 10 kD c ut-off filter
LC-MS/MS analysis
Fig.1.FlowchartfortheLC–MS/MSquantificationofWRNexonucleaseactivity.
able to degrade the complementary strand as efficiently as evaluatedbyanalyzingthereleaseoffreedC(Fig.3B).Insummary, themethoddescribedhereinopensupnewpossibilitiestostudy substrate and strand specificity of WRN in particular and exonucleasesingeneral.
Inconclusion,wehavedevelopedanovel,non-radioactiveLC–
MS/MS-basedquantitativemethod toassessWRNexonuclease function and activity with high sensitivity, accuracy, and precision.Asmassspectrometryisacommontoolinmolecular bioscience, this method represents a reliable and easy-to-use alternativetoexistingtechniqueswith50 end-labeledoligonu- cleotides. Our method is comparable to existing radiometric techniquesintermsofworkloadandeconomicaspects,butcanbe performedinhigherthroughputandenablesmassspectrometric structuralcharacterizationandfullquantificationoftheexonu- cleasedigestionproducts.Forthisreasonthisnewmethodshould beinstrumentalforseveralapplications.Asdemonstratedinthis study,theLC–MS/MSmethodcanbeusedtostudyexonuclease activityofWRN mutants(Fig. 2C),modulatory effects ofWRN interactionpartners andposttranslational modifications onits exonucleaseactivity(Fig.2D),andWRN’ssubstrateandstrand specificity (Fig. 3). Moreover, the LC–MS/MS method can be
employed to analyze the selectivity and processivity of WRN exonuclease with high specificity. For example, as shown by classicalradiometrictechniques,WRNexonucleaseisselectively blockedbyspecificbasemodificationssuchas8-oxo-guanine,8- oxo-adenine, and cholesterol adducts, but is active on other lesions such as uracil or hypoxanthine (Bukowy et al., 2008;
Machwe et al., 2000). Since released base adducts can be unequivocallyidentified,characterized,andquantifiedbymass spectrometry, the current method represents a promising approachtoextendsuchstudies.Inaddition,thismethodallows the screening of small molecule libraries for potential WRN exonucleaseinhibitors.InanalogytoarecentlyidentifiedWRN helicaseinhibitor(Aggarwaletal.,2011),suchpotentiallyexisting exonucleaseinhibitorscouldbeusefultoolstostudythefunction of WRN in a cellular context and may also have therapeutic potentialin cancertreatment.Wearecurrently planningsuch screeningapproachesforfindinginhibitorsorstimulatorsofthe WRNexonucleaseactivityatnanomolarlevels.Finally,theLC–
MS/MS-basedmethodcanbeextendedandadaptedtostudythe activity,specificity,andprocessivityofnucleasesotherthanWRN.
ExperimentswithEscherichiacoliExoIshowthefeasibilityofsuch extendedapplications(Fig.4).
0 20 40 60
0 50 100 150 200
R
2=0.960 4
Time [min]
A m ou nt dG [ fm ol ]
A B
C
0 100 200 300 400 500 600 700
0 20 40 60 8010 0 PDE po sitive cont.
WRN
R
2=0.926 3 C
WRN [nM]
A m ou nt dG [ fm ol ]
0 50 100 150 200 250
WRN [nM]
PAR P1 [nM]
- 40
40 -
40 40
40 80
***
***
n.d.
Amount dG [fmol]
D
WT-WRN X-WRN 0
100 200
300 ***
Amount dG [fmol]
Fig.2.AnalysisofWRNexonucleaseactivitybyisotopedilutionmassspectrometry.WRNdegradesanoligoduplexsubstratemimickingthetelomericrepeatsequence (TTAGGG)4tomononucleotides.Duetotherepetitivesequenceoftheoligoduplextheamountoffree20-deoxyguanosine(dG)correlateswiththeextentofoligoduplex degradation.PriortoLC–MS/MSanalysis,nucleotidesweredephosphorylatedusingalkalinephosphatase.SamplesweresubjectedtoLC–MS/MSanalysismonitoringthe transitionm/z268!152(dG)inthemultiplereactionmonitoring(MRM)mode.15N-labeleddGwasaddedasaninternalstandarddirectlyafterthereactiontoaccountfor technicalvariabilityduringsamplework-upandLC–MS/MSanalysis;theiontransitionm/z273!157wasmonitoredinMRMmode.(A)TimecourseanalysisofWRN exonucleaseactivityusing40nMWRNshowsthatthereactionreachedaplateauafter30min.(B)WRNdegradestheoligoduplexinaconcentrationdependentmanner.After incubationwith60–100nMWRNfor45min,thereactionreachedaplateau.Incubationoftheoligoduplexwithsnakevenomphosphodiesterase(PDE)servedasapositive control.(C)AmutationintheWRNexonucleasedomain(WRN-E84A,X-WRN)ledtoastronglydiminishedexonucleaseactivity(40nMofWT-WRNandX-WRN, respectively;reactionswererunfor45min)(D)RecombinantPARP1inhibitsWRNexonucleaseactivity.ConcentrationsofWRNandPARP1wereusedasindicated;reactions wererunfor45min.Oligoduplexsubstratewasusedinaconcentrationof75fmol(A,B,D)or100fmol(C)perreaction.Statisticalanalysiswasperformedusingone-way ANOVAfollowingBonferroni’smultiplecomparisontest.***p<0.0001.DatarepresentmeansSEM(N3);Rrepresentsthenon-linearregressioncoefficient.n.d.,not detectable.
AlimitationoftheassayincludesthataccesstoanLC–ESI-MS/
MSsystemisaprerequisiteforitsuse.Moreover,itislimitedto studyexonucleaseactivityinnon-cellularsystemsandsofaritis notapplicabletostudytheexonucleasedirectionality.
Insummary,theLC–MS/MS-basedexonucleaseactivityassay addstothearmamentariumofmethodstostudyDNAmetabolism ingeneraland,asdemonstratedinthisstudy,inparticularinthe fieldofmolecularagingresearch.
Acknowledgments
This work was supported by the Deutsche Forschungsge- meinschaft (Research Training Group[RTG] 1331 and Konstanz ResearchSchoolChemicalBiology,KoRS-CB)andtheUniversityof Konstanz (Ausschuss fu¨rForschungsfragen). SVissupported bya fellowship of the RTG 1331. OP and RM were supported by fellowshipsoftheKoRS-CB.Theworkwaspartiallysupportedby
theintramuralProgramoftheNationalInstituteonAging,National InstitutesofHealth.WewouldliketothankPeterC.Dedon,ErinG.
Prestwich, and Koli Taghizadeh from the MIT Center for Environmental Health Sciences for sharing their expertise in quantitativemassspectrometry.
AppendixA. Supplementarydata
Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.mad.2012.06.005.
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0 5 10 15 20 25 0
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R
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Exo I [ac tivity units]
Am ou nt d G [fm o l]
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