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ProcNatlAcadSciUSAVol95pp1057010575September1998BiochemistryIdimerizat


ThetypeIIsrestrictionendonucleaseIisolatedfromvobacteriumokeanokoitesrecognizesanasymmetricnucleotidesequenceandcleavesbothDNAstrandsoutsideoftherecogni-tionsite51Thegeneshavebeenclonedandsequenced23T

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Document on Subject : "ProcNatlAcadSciUSAVol95pp1057010575September1998BiochemistryIdimerizat"— Transcript:

1 Proc.Natl.Acad.Sci.USAVol.95,pp.10570±1
Proc.Natl.Acad.Sci.USAVol.95,pp.10570±10575,September1998BiochemistryIdimerizationisrequiredforDNAcleavage*,DAVIDA.WK.AGGARWAL*NewEnglandBiolabs,Inc.,32TozerRoad,Beverly,MA01915;andStructuralBiologyProgram,DepartmentofPhysiologyandBiophysics,Box1677,MountSinaiSchoolofMedicine,1425MadisonAvenue,NewYork,NY10029EditedbyRobertT.Sauer,MassachusettsInstituteofTechnology,Cambridge,MA,andapprovedJune22,1998(receivedforreviewApril7,1998)ABSTRACTIisatypeIIsrestrictionendonucleasecomprisedofaDNArecognitiondomainandacatalyticdomain.ThestructuralsimilarityoftheIcatalyticdomaintothetypeIIrestrictionendonucleaseHImonomersuggestedthattheIcatalyticdomainsmaydimerize.Inaddition,thestructure,presentedinanaccompanyingpaperinthisissueofProceedings,revealsadimerizationinterfacebetweencatalyticdomains.WeprovideevidenceherethatIcatalyticdomainmustdimerizeforDNAcleavagetooccur.First,weshowthattherateofDNAcleavagecatalyzedbyvariousconcentrationsofarenotdirectlyproportionaltotheproteinconcentration,sug-gestingacooperativeeffectforDNAcleavage.Second,wecon-structedaIvariant,N13Y,whichisunabletobindtheIrecognitionsequencebutwhenmixedwithwild-typeincreasestherateofDNAcleavage.Additionally,thecatalyticdomainthatlackstheDNAbindingdomainwasshowntoincreasetherateofwild-typeIcleavageofDNA.WealsoconstructedanIvariant,D483A,R487A,whichshouldbedefectivefordimerizationbecausethealteredresiduesresideattheputativedimerizationinterface.Consistentwiththedimerizationmodel,thevariantD483A,R487ArevealedgreatlyimpairedDNAcleavage.Basedonourworkandpreviousreports,wediscussapathwayofDNAbinding,dimerization,andcleavagebyIendonuclease. ThetypeIIsrestrictionendonucleaseI,isolatedfromvobacteriumokeanokoites,recognizesanasymmetricnucleotidesequenceandcleavesbothDNAstrandsoutsideoftherecogni-tionsite:5(1).Thegeneshavebeenclonedandsequenced(2,3).Theendonucleaseconsistsof587aawithamolecularmassof65.4kDa(2,4).Ihasbeenshowntoexistasamonomerinsolution,basedongelfiltrationandsedimentationexperiments(4).Ithasbeenconcludedthat,whenboundtoDNAandanalyzedbygel-mobilityshiftexperiments,onlyonemonomerofIwasboundtoitsDNArecognitionsequence(5).Thegel-mobilityshiftexperiments,wherea1:1complexwasobserved(5),wereperformedwithaIpre-cleavedDNA.ThereforethiscomplexrepresentsIboundtotheDNAproductnottotheDNAsubstrate.I,thetypicaltypeIIrestrictionendonucleases,suchRIorRV,formatighthomodimerinsolutionandbindtoDNAasahomodimer.Eachmonomericsubunitofho-modimercontainsonecatalyticcenter.MutationalanalysisbyWaughandSauer(6)suggeststhatthereisasinglecatalyticcenterperImonomer.ThisledthemtoconcludethateitherImustrearrangeitscatalyticcenterforsequentialcleavageofeachDNAstrandoritmustformahigherordercomplextocleavebothstrandsofDNA(6).Basedonproteolyticstudies,itwasshownthatIendonu-cleasecontainstwoseparatestructuraldomains,oneforDNArecognitionandoneforDNAcleavage(7).Apurified41-kDaN-terminalproteolyticfragmentboundtherecognitionsequencespecificallybutdidnotcleaveDNA,whereasthe25-kDaC-terminalproteolyticfragmentinthepresenceofMgDNAnonspecifically(7).MutationalanalysisfurthersupportedamodularstructureofI.AC-terminaldeletionvariantofIshowedthesameDNAbindingpropertiesasthewild-typeenzyme(8).Ontheotherhand,asingleaminoacidsubstitutionintheC-terminalhalfoftheIproteindisabledendonucleaseactivitybutdidnotaffecttheabilityoftheenzymetobindspecificallytoDNA(6).OtherstudieshavedemonstratedthatIcatalyticdomaincanbefusedtoDNAbindingproteinstoyieldcatalyticallyactivechimericproteinswithnovelrecognitionspecificities(9±12).YonezawaandSugiura(13),byusingfourdifferentfootprint-ingtechniques,showedthatIinteractswiththetargetrec-ognitionsequencefromthemajorgroovesideoftheDNAhelixandthatDNAprotectionatthesiteofcleavageintheabsenceofdivalentmetalionisveryweak.WaughandSauer(6)usedDNasefootprintingandmethylationprotectionwithcleavage-deficientvariantsofIinthepresenceofdivalentmetaliontoshowalsoaweakprotectionpatternaroundthesiteofcleavage(6).ThethreedimensionalstructureofIcomplexedtoDNAconfirmedthattheproteiniscomprisedoftwofunctionaldo-mains,aN-terminalDNArecognitiondomainandaC-terminalcatalyticdomain(14).Together,theIstructureinthepres-enceofDNA(14)andintheabsenceofDNA(15)providesabasisforthebiochemicalandmutationalstudiesreportedhere.ThestructureinthepresenceofDNAshowedasinglemoleculeapproachingDNAfromthemajorgrooveside,withtherecognitiondomainmakingallofthebase-specificcontactsattherecognitionsite.ThecatalyticdomainofIrevealedastruc-tureremarkablysimilartoamonomerofthetypeIIrestrictionHI(14±16).ThreecatalyticallyimportantresiduesofI(Asp-450,Asp-467,andLys-469)superimposewiththecatalyticresiduesAsp-94,Glu-111,andGlu-113ofHI.Therefore,whetherexaminedbymutationalanalysis(6)orbystructuralcomparison(14),thereappearstobeonlyonecatalyticsiteperImolecule.Thisraisesthequestion,``HowdoesmonomericIcleavebothDNAstrandsbyusingasinglecatalyticcenter?''TheIstructureshowedthatthecatalyticdomainissequesteredalongsideoftherecognitiondomainanddoesnotcontactDNAatthecleavagesite,inaccordancewiththeearlierfootprintingstudies(6,13).Fromthestructure,thecatalyticdomaincanberelocatedatthesiteofcleavagebyasimplerotationaroundthelinkersegmentsothatthecatalyticresiduesarepositionedinthevicinityofthescissilephosphodi-esterbondlocated13ntawayfromtherecognitionsite(14).However,forthesamecatalyticdo

2 maintocleavetheotherDNAstrand,itwouldreq
maintocleavetheotherDNAstrand,itwouldrequiretheenzymetoadoptasecondconfigu-ration,whichisdifficulttomodelwithoutextensiverefolding(14). Thepublicationcostsofthisarticleweredefrayedinpartbypagechargepayment.Thisarticlemustthereforebeherebymarked``advertisement''inaccordancewith18U.S.C.§1734solelytoindicatethisfact.1998byTheNationalAcademyofSciences0027-8424PNASisavailableonlineatwww.pnas.org. Thispaperwassubmitteddirectly(TrackII)totheProceedingsoffice.Abbreviations:FokN13Y,variantofIendonucleasecontainingAsn13Tyrsubstitution;IcatalyticdomainrepresentingC-terminal196aaoftheendonuclease;D450A,catalyticallyinactivecatalyticdomaincontainingAsp-450Alasubstitution;D483A,R487A,variantofIendonucleasecontainingAsp-483AlaandArg-487Alasubstitutions.Towhomreprintrequestsshouldbeaddressed.e-mail:schildkr@neb.com. Inthispaper,weprovideevidencethatIdimerizationisrequiredforDNAcleavage.EXPERIMENTALPROCEDURESBacterialStrainsandPlasmids.EscherichiacolistrainER2566ER2566fhuA2,lacZT7gene1,(mcrC-mrr)114IS10,R(mcr-73miniTn10±TetS)2,endA1]containingachromosomalcopyoftheT7RNApolymerasegenewasconstructedbyE.Raleigh(NewEnglandBiolabs)andusedforexpressionofIvariants.PlasmidpAFP1containingtheclonedIendonucleasegene(2)andplasmidpAFD2containingtheImethyltransferasegene(2)wereconstructedbyW.E.Jack(NewEnglandBiolabs).ThepAFP1derivativecodingforthecatalyticallyinactiveD450AvariantofI;theplasmidpSKFok1,aderivativeofpBlue-),containingasingleIsite;andtheplasmidpSKFok0,thederivativeofpSKFok1inwhichdeletionoftheIIfragmentremovedthesingleIrecognitionsequencewereconstructedbyC.Noren(NewEnglandBiolabs;unpublishedwork).pTYB4andpTYB2areproductsofNewEnglandBiolabs.SubcloningoftheIGeneintotheImpactOne-StepProteinPurificationSystem.ThefulllengthIendonucleasegeneandthetruncatedgenecodingonlyforthecatalyticdomainofendonucleasewereamplifiedbyPCRbyusingplasmidpAFP1asatemplate.The5primer,5-AGCCCATGGTTTCTAAAAT-ACGTACGTTCGGTTGGG-3,foramplicationofthefullIendonuclease,wascomplementarytotheexceptforsilentmutationsinthe6thand7thcodonstocreateaWIsite.Theprimer,5-GTGCATATGCAACTAGT-CAAAAGTGAACTGG-3,foramplificationofthecatalyticdomainofIwasflankedbytheIsite.Therestoftheprimersequencematchedthegenesequencestartingfromthe384thcodon.The3reverseprimer,5CAAAGTTTATCTCGCCGTTATTAA-3,iscomplementarytotheendofgeneandisflankedbytheIsite.TheresultingPCRproductswereagarose-gelpurifiedandcleavedwithrestrictionendonucleases:IandIinthecaseofthefulllengthIgene;orIandIinthecaseofthegenecodingfortheIcatalyticdomain.Thegel-purifiedrestrictionfragmentcontainingthefulllengthwasligatedintotheIandIdigestedvectorpTYB4.Thiscreatedthecon-structpTYB4FokR(Fig.1)wheretheCterminusofthegenewasfuseddirectlytotheinteinoftheImpactPurificationSystem(17,18).Thegel-purifiedrestrictionfragmentcontainingcatalyticdomaincodingsequencewasligatedintotheIandIdigestedvectorpTYB2.TheresultingconstructwasdesignatedpTYB2FokCD.NosequencealterationsotherthandesiredmutationsweredetectedbyDNAsequenceanalysis.ConstructionofCatalyticallyInactiveIVariants.IfragmentofpTYB4FokRwasreplacedbythesamefragmentfromthepAFP1D450Aconstruct(seeabove)tocreatetheplasmidderivativepTYB4codingforacatalyticallyinactiveIvariant.Similarly,theIfragmentofpTYB2FokCDwasreplacedbyIfragmentfrompAFP1D450AtocreatepTYB2ConstructionofaBinding-DeficientIVariant.TocreateaIvariant,Asn13wasreplacedbyabulkyaminoacid,tyrosine.Thefollowingmutagenicprimerswereusedtoamplifythe0.29-kbIfragmentoftheIgene:-ATACGTACGTTCGGTTGGGTTCAATATCCAGGTA-ATTTGAG-3(introducesN13Y)and5-GCCCAACGCAAA-AAACCGTCAGATGA-3ThePCRproductwascleavedwithWIandIrestrictionendonucleases,andthegel-purifiedrestrictionfragmentwasligatedintotheWIandaIdigestedpTYB4FokRvector.Thiscreatedanewderivative,pTYB4FokN13Y,codingfortheIvariantwithalteredbase-specificcontacts.ThesequenceofthereplacedDNAfragmentwasverifiedbyDNAsequenceConstructionofDimerization-DeficientIVariant.IgenesegmentflankedbyIandGIrestrictionsiteswasamplifiedbyPCRbyusingpTYB4FokRasatemplateandamutagenicprimerthatintroducedsubstitutionsD483AandR487A,5-CAATTGGCCAAGCAGCTGAAATGCAAG-CATATGTCGAAGAAAATCAAACACG-3.ThesecondprimermatchedtheDNAsequencedownstreamtheandwasflankedbyGIrestrictionsite5-ACGCTGTACAT-AGTTTCTCTTCC-3.TheGIfragmentofpTYB4FokRwasreplacedwithIandGIdigestedmutagenicPCRproduct.Thiscreatedanewderivative,pTYB4whichcodedforadimerization-deficientIvariant.TargetProteinExpressionandPurification.Wild-typerestrictionendonucleasewaspurifiedtohomogeneityasde-scribedearlier(19).Theconcentrationofwild-typeIproteinwasdeterminedspectrophotometricallyat280nmbyusingtheextinctioncoefficientof72,520MTheOne-StepProteinPurificationSystem(Impact)(17)wasusedtofacilitatepurificationofD450A,andD483A,R487Avariants.Thistechniqueisbasedontheself-catalyzedcleavageoftheinteinthatcanbecontrolledinvitrobyDTT(18).Thechitinbindingdomainisfusedgenet-icallytotheCterminusoftheinteinandservesasanaffinitytagtobindI±inteinfusionontoachitincolumn.TheIisreleasedfromthefusionaftertheinductionoftheinteincleavagereactionbyadding50mMDTT,whileintein±chitinbindingdomainfusionremainsboundtothecolumn(17).E.coliER2566cellscontainingmutantpTYB-Fokconstructsweregrownat37ÉCinLuria±Bertanimediumsupplementedwith0.1mgmlampicillin.Expressionofthetargetproteinwasinducedbyaddingisopropyl-thiogalactosidetoafinalcon-centrationo

3 f0.1mMatmid-logphaseofgrowth(KlettAfteri
f0.1mMatmid-logphaseofgrowth(KlettAfterinduction,thecultureswereincubatedovernightat15ÉC.Thecellswereharvested,resuspendedincolumnbuffer(20mMpotassiumphosphate,pH7.40.1mMEDTA0.5MNaCl)anddisruptedbysonication.Thecrudeextractcontainingfusionproteinwasloadedontoachitincolumn(NewEnglandBiolabs)andwashedwithtwocolumnvolumes,andthecolumnwasflushedquicklywith2columnvolumesof20mMpotassiumphosphatebuffer(pH7.4),0.1mMEDTA,and50mMNaClcontaining50mMDTT.Thecolumnwasincubatedat4ÉCovernighttoinduceself-cleavageoftheproteinfusion(17).Next,thetargetproteinwaselutedfromthecolumnwithcolumnbufferanddialyzedtoremoveexcessDTT.Thepurifiedproteinswereconcentratedbydialysisagainst20mMpotassiumphosphate(pH7.4),0.1mMEDTA,50mMNaCl,and50%glycerolandstoredIvariantspurifiedviatheImpactSystemhavetwoadditionalglycineresiduesattheircarboxyltermini.DNACleavageAssays.Thecleavageactivityofmutantvariantswasassayedbyincubatingvariousamountsofproteinat37ÉCineither30or50lofstandardIreactionbuffer(20mM .1.PlasmidmapofthepTYB4FokR.ThegeneisplaceddownstreamoftheT7promoter,andisin-framewiththeendoftheinteincodingsequence.CBD,chitinbindingdomain.Biochemistry:Bitinaiteetal.Proc.Natl.Acad.Sci.USA95(1998) Tris-acetate,pH7.910mMmagnesiumacetate50mMpotas-siumacetate1mMDTTg/mlBSA)containing1goforplasmidpSKFok1DNA.Thereactionwasterminatedbyadditionof10lofstopsolution(60mMEDTA,pH8.01%SDS0.02%bromphenolblue),andreactionprod-uctswereanalyzedbyelectrophoresisin1%agarosegels.ForquantitativeIactivityassays,plasmidpSKFok1wasfirstlinearizedwiththerestrictionendonucleaseItomakealinearsubstratewiththeIrecognitionsitepositionedinthecenteroftheDNAmolecule.DNAthenwaspurifiedbyphenol-chloroformextractionfollowedbyisopropanolethanolprecipi-tation.DNAconcentrationwasdeterminedspectrophotometri-callyat260nm.Iwasaddedtoeither70or200lofreactionbuffer(seeabove)containing6.3nMsubstrateDNA.Sampleswereincubatedat37ÉC,10-laliquotswereremovedatvarioustimeintervals,andthereactionproductswereanalyzedbyagarosegelelectrophoresis.Theethidiumbromide-stainedgelsweredigitizedwithIS-500DigitalImagingSystem(AlphaInno-tech,SanLeandro,CA).TheintensityoftheproductbandwasquantitatedbyusingtheNIHIMAGEprogramv.1.61.RESULTSRateofCleavagevs.EnzymeConcentration.TherateofDNAcleavagecatalyzedbyvariousIconcentrationswasdeter-minedtoestablishtherelationshipbetweentheinitialvelocityofthereactionandtheenzymeconcentration.Linearizedplasmid1DNAcontainingasingleIrecognitionsiteposi-tionedinthecenteroftheDNAmoleculewasusedasasubstrate.UsingsuchaDNAsubstratesimplifiedtheevaluationoftheconcentrationofreactionproductsthat,becauseoftheirsamesize,runontheagarosegelasasingleband.AlimitingamountofIrestrictionendonuclease(from0.1to1.0nMfinalconcentration)wasaddedlasttothereactionmixture,whichwaspreheatedat37ÉC.Theprogressofeachreactionwasmonitoredbywithdrawing10-lsamplesattimedintervals.Typicalresultsofsuchcleavageassays,performedwith0.2and0.6nMI,areshowninFig.2.TheamountofcleavedDNAwasexpressedinarbitraryunitsafterthequantitativeevaluationoftheintensityoftheproductbandinanethidiumbromide-stainedagarosegel.TheplotsshowingtheprogressoftheDNAproductformationwithtimeatdifferentIconcen-trationsarepresentedinFig.3.Theseplotswereusedtocalculatetheinitialvelocity((P]ydt)ofeachreactioncatalyzedbyaIconcentration.Whenmeasuredvaluesofplottedagainstthecorrespondingvaluesof[I]anonlinearplotwasobtained(Fig.3,).Thisindicatedthat,atlowconcentrations,theinitialvelocityofthereactionisnotdirectlyproportionaltotheenzymeconcentration,suggestingthattheI-catalyzedreactionishigherthanfirstorderwithrespecttotoFokI].Amongthepossibleinterpretations,thenonlinearrela-tionshipofvs.[I]canbeexplainedbyacooperativebindingoftwoImoleculestocleavebothstrandsofDNA.CharacterizationofN13YVariant.IfthecatalyticallyactiveIcomplexrequirestwoImolecules,i.e.,twocatalyticdomains,fordouble-strandedDNAcleavage,thenthereactionatlowconcentrationsofIproteinmaybestimulatedbyabinding-deficientvariantofIthatcontainsanactivecatalyticdomain.Thethree-dimensionalstructureoftheIendonu-cleaseboundtotheDNAsubstraterevealedthatAsn13makesbidentatehydrogenbondswiththecentraladeninebaseoftherecognitionsequenceGGATG(14).Substitutionofthisresiduewiththebulkyaminoacidtyrosineshoulddestroybase-specificcontactsattherecognitioninterface.SuchamutantproteinwouldbeincapableofbindingtothetargetDNAandthereforewouldbeimpairedforDNAcleavage.Totestthis,thepTYB4FokN13YderivativewasconstructedasdescribedinExperimentalProcedures.ThemutantN13Yproteinthenwasexpressedandpurifiedbyasinglechromatographicstepfromachitincolumn.Thepurifiedproteinwas95%pureasdeterminedbyN13Y(200nM)wasincubatedwithsupercoiledandlinearizedpSKFok1DNAfor1hat37ÉC.Atthesecondi-tions,nodetectabledouble-strandorsingle-strandcleavageofDNAsubstratewasobserved.StimulationofIActivitywithTotesttheN13YvariantforitsabilitytostimulateIrestrictionendonuclease,thesubstrateDNAwasincubatedwitheither1nMIalone,500nMN13Yproteinalone,orthepremixedI(1nM)andN13Y(500nM)proteincombination.Fig.revealsthattheDNAcleavagereactioncatalyzedby1nMIaloneisslow.After12minonlyasmallportion(5%)ofthesubstrateDNAiscleaved.Underthesamereactioncondi-tions,500nMN13YdidnotshowanydetectableDNAcleavageactivity(Fig.4).However,whenIendonucleasewassupplementedwith500nMN13Yprotein,a10±20-foldincreaseintherateofDNAcleavagew

4 asobservedrelativetoIalone(Fig.4).Thissu
asobservedrelativetoIalone(Fig.4).Thissuggeststhatwild-typeIproteininteractswiththeN13Ymoleculetofacilitatesite-specificCharacterizationofCDandD450AVariants.pTYB2FokCDandpTYB2D450AderivativescodefortheC-terminal196aaofIrestrictionendonuclease,constitutingthe25-kDacatalyticdomain.Inaddition,thederiv-ativepTYB2D450AcodesforacatalyticallydefectiveCDprotein,D450A.ThepurifiedCDandD450Aproteinsweretestedfortheabilitytocleave .2.TimecourseoftheI-catalyzedDNAcleavagereaction.LinearizedpSKFok1DNA(6.3nM)wasincubatedat37ÉCwith0.2(or0.6nM(Irestrictionendonucleasein200lofreactionbuffer(20mMTris-acetate,pH7.910mMmagnesiumacetatemMpotassiumacetate1mMDTTg/mlBSA).Atthetimepointsindicated,10-lsampleswerewithdrawn,immediatelyquenchedbyadding5lofstopsolution,andsubjectedtoelectro-phoresison1%agarose-gel. .3.RateofDNAcleavagewithvariousIconcentrations.TheconditionsofDNAcleavagereactionaredescribedinFig.2.Sampleswerewithdrawnattheindicatedtimesfromthereactionsthatcontained0.1nM),0.2nM),0.4nM),0.6nM),0.8nM),and1.0nM).TheamountofcleavedDNAwasdeterminedasinExperimentalProcedures)Initialvelocityvs.Iconcentration.10572Biochemistry:Bitinaiteetal.Proc.Natl.Acad.Sci.USA95(1998) DNA.WhenCDproteinwasincubatedwith1nMDNAfor1hat37ÉC,therewasnodetectableDNAcleavage.AtCDconcentrations,nonspecificDNAcleavagewasobserved.Incontrast,incubating2.5D450Aaslongas20hwith1nMDNAat37ÉCdidnotshowanydetectableDNAcleavage.StimulationofIActivitywiththeCatalyticDomain.ulationofIwiththeCDproteinwasperformedasdescribedfortheN13Yvariant.After12min,1%ofDNAsubstratewascleavedby0.5nMIalone(Fig.4).NodetectableDNAcleavagewasobservedwith500nMalone(Fig.4).When0.5nMIwassupplementedwith20nMCD,noincreaseintherateofDNAcleavagewasobserved(datanotshown).However,with0.5nMIplus500nM50%ofsubstrateDNAwascleavedafter8min(Fig.).Thisrateis100-foldhigherthanwith0.5nMIalone.Incontrast,thecatalyticallyinactiveCDvariantD450AfailedtoactivateI(Fig.4).Theseresultsindicatethattwoseparatecatalyticdomainsarenecessaryforsite-specific,double-strandDNAcleavagebyDimerization-DefectiveIVariantDisplaysDiminishedCleavageofBothDNAStrands.InanaccompanyingpaperinthisissueofProceedings(15),wereportthestructureofIintheabsenceofDNA.Thestructurerevealsprotein±proteininterac-tionsbetweentwocatalyticdomainsthatmimicaHIho-modimer.CloseinspectionofthisinterfaceshowsthataminoacidsAsp-483andArg-487arelocatedwithinhydrogenbonddistancewiththecorrespondingresiduesintheothercatalyticdomainsubunit.Theseresidueswereconsideredtobegoodcandidatesfordisruptingthedimerinterfacewhilenotaffectingfoldingoftheproteinforthefollowingreasons:()theresiduesarelocatedonthesurfaceofthedomain;and()theseresiduesdonotcontactneighboringaminoacidresidueswithinthesamesubunit.ThepTYB4FokD483A,R487AconstructwasmadeandtheproteinwasexpressedandpurifiedasdescribedinExperimentalProcedures.CleavageofsupercoiledpSKFok1plasmidbyendonucleaseandD483A,R487Aproteinwascompared.AsshowninFig.5,after1hat37ÉC,6nMIcompletelyconverted20nMsupercoiledpSKFok1plasmidDNAtoafulllengthlinearproduct.IncubationofIwithaplasmidDNAthatdoesnotcontainaIrecognitionsequence,pSKFok0,showedthatIcleavagereactionissequence-specific(Fig.5Incontrasttothewild-typeendonuclease,theD483A,R487AvariantshowednodetectableDNAcleavageat9nM(Fig.5Ata10-to80-foldhigherconcentrationofD483A,R487Avariant,themajorproductobservedwastheopencircularformofplasmidDNA.Thenickingactivitywassite-specificbecauseD483A,R487AvariantdidnotconvertplasmidpSKFok0DNAtotheopencircularform(Fig.5).Theresultsindicatethatdouble-strandcleavageactivityoftheD483A,R487Avariantisreducedthreeordersofmagnitudeascomparedwiththedouble-strandcleavageactivityofwild-typeenzyme(Fig.5TypicaltypeIIrestrictionendonucleasesexistinsolutionashomodimersandareknowntobindtoandcleaveDNAashomodimers.Themolecularmassofthemonomersaregenerallyinthe20-to40-kDarange.Eachmonomericsubunitisrespon-sibleforhalfoftheDNAbase-specificcontacts,andeachsubunitcontainsonecatalyticcenterthatcleavesonestrandoftheduplex.Thesitesofcleavagearealwaysfoundsymmetricallypositionedwithintherecognitionsequence.Thethree-dimensionalstruc-turesofthetypeIIendonucleasesshowthattherecognitionandcatalyticfunctionsarewellintegratedintoasingleproteindomain(21).Ontheotherhand,thetypeIIsrestrictionendonucleaseIisalarger,65-kDaprotein.Itismonomericinsolution(4).TheasymmetricrecognitionsequenceandsitesofcleavageareseparatedbyoneturnoftheDNAhelix.TheDNArecognitionfunctionandsinglecatalyticcenterofIresideontwoseparatedomainsoftheprotein.HowdoesIcleavebothstrandsoftheUnlikethetypeIIendonucleases,thespecificrecognitionofDNAduplexbyIismediatedclearlybythesinglerecognitiondomain(14).However,thenatureoftheterminiofI-cleavedDNAiscomparabletotheterminiofHIorRIcleavedDNAbecauseallthreeendonucleasescleavedouble-strandedDNAtoleavea4-base,5-extension.Thethree-dimensionalstructuresofHIandRIshowasimilarcommoncoreandpositioningofthecatalyticresiduesrelativetothetargetedscissilephosphategroups(22).Thestructuralorga-nizationoftheIcatalyticdomaindisplaysastrikingsimilaritytothemonomerofHI(14).Thetwostructuressharea-sheetcoresurroundedby-helices.Theactivesite .4.StimulationofIendonucleaseby(N13Y,(CD,or(D450A.Reactionswereperformedat37ÉCin70of20mMTris-acetatebuffer(pH7.9),10mMmagnesiumacetate,50mMpotassiumacetate,1mMDTT,100mlBSA,and6.3nMlinearizedpSK

5 Fok1DNA;10-lsampleswerewithdrawnattheind
Fok1DNA;10-lsampleswerewithdrawnattheindicatedtimesfromthereactionsthatcontained:()either500nMN13Y,1.0nMor1.0nM500nMN13Y;()either500nMCD,0.5nMI,or0.5nM500nMCD;and()either500nMD450A,1.0nMI,or1.0nM500nMD450A.Sampleswerequenchedbyadding5lofstopsolutionandwereanalyzedbyelectrophoresison1%agarosegel.Biochemistry:Bitinaiteetal.Proc.Natl.Acad.Sci.USA95(1998) residuesofbothendonucleasesoccuratoneendoftheandaresuperimposedeasilyoneachother(14).Thisdata,togetherwiththestructuralorganizationofIintheabsenceofDNA(15),ledustoproposethatIendonucleasemayuseastrategysimilartoRIandHIforDNAcleavage.Fromthisperspective,wehavepostulatedthat()twoImoleculesareinvolvedindouble-strandedDNAcleavage;()DNAcleav-agereactionbyIproceedsviaanenzymedimerizationstepthattakesplaceinthepresenceofboththespecificDNAanddivalentmetalion;and()dimerizationoccursattheinterfaceoftwocatalyticdomainsofMeasurementoftherateofDNAcleavageatdifferentenzymeconcentrationssuggeststhattheIcatalyzedreactionishigherthanfirstorderwithrespecttoI.ThisisconsistentwiththecooperativebindingoftwoImolecules.Ontheotherhand,thenonlinearrelationshipbetweentheinitialvelocityofthereactionandIatlowenzymeconcentrationsalsocouldbeexplainedbyotherfactors;forexample,atlowproteinconcen-tration,theenzymemayfirstbeadsorbedtothesideofthereactionvessel.AlthoughthereactionbufferusedintheDNAcleavageexperimentscontained100mlBSA,whichshouldalleviatesuchundesirablenonspecificbindingofI,wecannotruleoutthatsomeanalogousinteractiondidnottakeplace.ToobtainadditionalevidenceforIcooperativity,variantswithmutated(N13Y)ordeleted(CD)recogni-tiondomainswereconstructedandwereshowntostimulateactivity.WhenincubatedalonewithDNA,thesevariantswereshowntobeincapableofcleavingDNA.However,whenN13YwasincubatedtogetherwithalimitingamountoftherateofDNAcleavagewasincreased10-fold.Likewise,inthepresenceofIactivityincreased100-fold.TheinactiveIcatalyticdomainD450Afailedtostimulateendonuclease.Furthermore,theinactiveIcatalyticdomaindidnotappeartoinhibitthelowlevelofIactivity,indicatingthattheinactivecatalyticdomainisincapableofformingacomplexwithI.AprecleavagecomplexprobablyreliesonthebindingenergyderivedfromcontributionsoftheappropriateionicinteractionsbetweenthemagnesiumcoordinatedwithresiduesD450,D467,andthescissilephosphate.Inthecatalyt-icallydefectivedomain,D450isreplacedbyalanine.Itisexpectedthatthiswillupsetthecoordinationofthemagnesium,hencethenetworkofinteractionsthataffordthetransientbindingenergytoformapre-cleavagecomplex.ThestimulationofIbyCDsuggeststhataImonomermustinteractwithasecondcatalyticcenterfordouble-strandedDNAcleavagetooccur.Thus,duringcleavagebywild-typeIendonuclease,twoImoleculesinteractwitheachothertoaligntwocatalyticcentersforthecleavageofbothDNAstrands.ThecooperativitythatIdisplaysforDNAcleavageisapparentataconcentrationrangebelow1nM.Above1nM,therateofDNAcleavageincreasesproportionatelytotheamountofIaddedtothereaction.ThissuggeststhattheequilibriumbindingconstantofIforaDNAcomplexis1nM.Inthestimulationexperiments,a100nMconcentrationofmutantIvariantisrequiredtoobservethesamerateofDNAcleavageas3nMIalone.Theseresultsindicatethatthefulllength,wild-typeImoleculeistwoordersofmagnitudemoreefficientatfacilitatingDNAcleavagethanthemutantproteinsthathadtheirDNArecognitiondomainmutated(N13Y)ordeleted(CD).ThissuggeststhatthesecondrecognitiondomainalsoplaysaroleintheformationandorstabilityofthecatalyticallyactiveIdimerDNAcomplex.Thephenomenonof``sequestration''oftheIcatalyticdomainbytherecognitiondomainthatwasobservedinthethree-dimensionalstructure(14)mayexisttopreventthecata-lyticdomainfrominteractingwiththeDNAwhilethemoleculescanstheDNAinsearchofanewrecognitionsiteorsearchesforanothermonomerthatalreadyhasboundtoarecognitionsequence.ThesequesteringofthecatalyticdomainmayexplainhowImanagestoregulateitscleavageactivity.ThecatalyticdomainremainssequesteredduringbindingtoitsDNArecognitionsequenceandistriggeredforreleasebydimer-ization.Whenwild-typeIisincubatedwithsupercoiledDNAatlowenzymeconcentrationandthereissufficientenzymetobindonlyasmonomer,itmaybeexpectedtoonlynicksuper-coiledDNA.ThiswouldresultintheaccumulationoftheopencircularformofDNA.However,theexperimentalresultsdonotsupportsuchamodelbutrathersupporttheviewthatthecatalyticdomainremainsinactive.Atamolarratioof1:20ofI:DNA,aslightexcessofthenickedintermediateoverthelinearDNAwasobserved.Ata1:8ratioofI:DNA,thelinearproductwaspredominantinthereaction(Fig.5).ThebehaviorofthedoublemutantD483A,R487Aalsosupportstheviewthatthecatalyticdomainisinactiveuntildimerizationoccurs.TheR487Amutationstargetedthecatalyticdomaindimer-izationinterfacewhileleavingtherecognitiondomainandcat-alyticcenteroftheenzymeunchanged.However,byalteringthecatalyticdomaindimerizationinterface,theIcleavageof .5.ComparisonofDNAcleavagebyIandD483A,R487A.Thecleavagereactionswereperformedfor1hat37ÉCin30lof20mMTris-acetatebuffer(pH7.9),10mMmagnesiumacetate,50mMpotassiumacetate,1mMDTT,and100mlBSA,containing20nMof)supercoiledplasmidpSKFok1or()supercoiledplasmidpSKFok0andeither(Ior(D483A,R487A.TheconcentrationsofIandD483A,R487Aareindicated.(CC),covalentlyclosedformofplasmidDNA;(OC),opencircleform;(L),linearform.10574Biochemistry:Bitinaiteetal.Proc.Natl.Acad.Sci.USA95(1998) eitherstrandoftheDNAduplexwasimpaired.Thus,neithertheresultsforIortheD48

6 3A,R487AvariantindicatethattheImonomerca
3A,R487AvariantindicatethattheImonomercancleaveoneDNAstrand.Morelikely,themonomercomplexedtothespecificDNAisinactivebecauseitscatalyticdomainissequestered.Alternatively,theIcatalyticdomainmaybeabletodissociatefromtherecognitiondomainaftertheImakesspecificcontactswithitsDNAsubstratebutmaynotbeabletoproperlyalignitscatalyticcenterforthephosphodiesterbondcleavagewithoutdimerformation.Basedonthisworkandpreviousreports,thefollowingviewofIendonucleaseemerges:Iexistsinsolutionasamonomer,andfirstbindstoDNAasamonomer.Thecomplexiscatalyticallyinactive.AsecondImonomerarriveseitherwhen:()DNAisscanneduntilthemonomercollideswiththefirstImolecule.Presumably,onlymonomersarrivinginoneoftwoorientationsatthe3endoftherecognitionsequencecandimerizesuccess-fully;()themonomericDNAcomplexinteractswithanothermonomericDNAcomplex;or(ImonomerfromsolutioncollideswithaDNAcomplex.Thisalterna-tiveseemstheleastlikelybecausethebinding-deficientmutantproteinsarerelativelypoorstimulatorsonamolarbasis.ThecatalyticdomainofeachImoleculeswingsawayfromtherecognitiondomaintopositionitscatalyticsitesoppositethetargetedphosphodiesterbond.Theresiduesatthecatalyticdomaindimerizationinterfaceinteractwitheachother,formingadimerthatresemblesastructurenotunlikeaprototypicaltypeIIrestrictionendonucleaseboundtoDNA.Theformationofthedimermayrequiremagnesiumion(s).CleavageofbothDNAstrandsoccursafterIdimerizationinthepresenceofmagnesium.ThedimerizationmodelofferstwolevelsofcontroltopreventIfromcleavingDNAnonspecifi-cally.First,thereleaseofthecatalyticdomainfromitsseques-teredpositionmaydependonsequence-specificDNAbinding.Second,thedimerizationofthecatalyticdomainmayberequiredtoachieveproperalignmentofthetwocatalyticdomainsforphosphodiesterbondcleavage.DimerizationinthepresenceofspecificDNAmaybeastrategyothertypeIIsrestrictionendonucleasesuse.Todate,alltypeIIsendonucleasesstudied(57I)aremonomersinsolution(23±26).IIbindstoitsrecognitionsequenceasamonomer(27).Severalchimericenzymeshavebeencreatedbyfusingthecatalyticdomainwithsite-specificDNAbindingdomains.catalyticdomainwasfusedgeneticallytoDrosophilaUbxodomain(9)andwithSp1-QNRandCP-QDRzincfingerproteins(10,11).Recently,aZ-DNA-specificnucleasewasconstructedfollowingthesamestrategy(12).However,therateandefficiencyofDNAcleavagebythehybridendonucleasesaremuchlowercomparedwiththewild-typeI(10).Itisdifficulttoachievecompletesite-specificcleavagewithhybridnucleasesbecausehighproteinconcentrationandorlongincubationtimesusuallyleadtoanonspecificDNAcleavage.ThedimerizationofIsuggeststhatcleavageratesandthespecificityofchimericendonucleasesmaybeimprovedbyaddingexcessexogenouscatalyticdomainorbyconstructingatriplefusionwheretheheterologousDNAbindingmotifislinkedtothecovalentlyfusedIcatalyticdomaindimer.WethankW.Jack,C.Noren,andX.XiongforprovidingplasmidsandIconstructs.WethankR.WhitakerforassistancewithmoleculardisplayprogramsandtechnicaladviceinselectingsubstitutionsinthattargetedDNArecognition,L.DornerandR.Kuceraforpurification,andM.-Q.XuandS.ChongforassistancewithImpactSystem.WearegratefultothepersonnelofNEBOrganicSynthesisandSequencingdivisionsforsyntheticDNAsandsequencingofnewDNAconstructs.WethankS.HalfordforhelpfuldiscussionsandR.Roberts,C.Noren,andR.Whitakerforcriticalcommentsonthemanuscript.TheworkinA.Aggarwal'slaboratorywassupportedbyNationalInstitutesofHealthGrantGM44006.1.Sugisaki,H.&Kanazawa,S.(1981)2.Looney,M.,Moran,L.S.,Jack,W.E.,Feehery,G.R.,Benner,J.S.,Slatko,B.E.&Wilson,G.G.(1989)3.Kita,K.,Kotani,H.,Sugisaki,H.&Takanami,M.(1989)J.Biol.4.Kaczorowski,T.,Skowron,P.&Podhajska,A.J.(1989)209±216.5.Skowron,P.,Kaczorowski,T.,Tucholski,J.&Podhajska,A.6.Waugh,D.S.&Sauer,R.T.(1993)Proc.Natl.Acad.Sci.USA9596±9600.7.Li,L.,Wu,L.P.&Chandrasegaran,S.(1992)Proc.Natl.Acad.Sci.USA2764±2768.8.Li,L.,Wu,L.P.,Clarke,R.&Chandrasegaran,S.(1993)79±84.9.Kim,Y.-G.&Chandrasegaran,S(1994)Proc.Natl.Acad.Sci.883±887.10.Kim,Y.-G.,Cha,J.&Chandrasegaran,S(1996)Proc.Natl.Acad.Sci.USA1156±1160.11.Huang,B.,Schaeffer,J.,Li,Q.&Tsai,M.-D.(1996)J.Protein481±489.12.Kim,Y.-G.,Kim,P.S.,Herbert,A.&Rich,A.(1997)Proc.Natl.Acad.Sci.USA13.Yonezawa,A.&Sugiura,Y.(1994)Biochim.Biophys.Acta369±379.14.Wah,D.,Hirsch,J.A.,Dorner,L.F.,Schildkraut,I.&Aggarwal,A.K.(1997)Nature(London)15.Wah,D.A.,Bitinaite,J.,Schildkraut,I.&Aggarwal,A.K.(1998)Proc.Natl.Acad.Sci.USA10564±10569.16.Newman,M.,Strzelecka,T.Dorner,L.F.,Schildkraut,I.&Aggarwal,A.K.(1995)656±663.17.Chong,S.,Mersha,F.B.,Comb,D.G.,Scott,M.E.,Landry,D.,Vence,L.M.,Perler,F.B.,Benner,J.,Kucera,R.B.,Hirvonen,C.A.,etal.18.Chong,S.,Yang,S.,Paulus,H.,Benner,J.,Perler,F.B.&Xu,M.-Q.(1996)J.Biol.Chem.22159±22168.19.Hirsch,J.,Wah,D.,Dorner,L.F.,Schildkraut,I.&Aggarwal,A.K.(1997)FEBSLett.136±138.20.WisconsinPackageVersion9.1(1997)(GeneticsComputerGroup,Madison,WI).21.Aggarwal,A.K.(1995)Curr.Opin.Struct.Biol.22.Newman,M.,Strzelecka,T.Dorner,L.F.,Schildkraut,I.&Aggarwal,A.K.(1994)Nature(London)660±664.23.Sektas,M.,Kaczorowski,T.&Podhajska,A.J.(1992)AcidsRes.433±438.24.Tucholski,J.,Skowron,P.&Podhajska,A.J.(1995)25.Shinomiya,T.,Kobayashi,M.&Sato,S.(1980)NucleicAcidsRes.26.Janulaitis,A.,Petrusyte,M.,Maneliene,Z.,Klimasauskas,S.&Butkus,V.(1992)NucleicAcidsRes.6043±6049.27.Sektas,M.,Kaczorowski,T.&Podhajska,A.J.(1995)Biochemistry:Bitinaiteetal.Proc.Natl.Acad.Sci.USA95(