Metal ion dependence of DNA cleavage by SepMI and EhoI restriction endonucleases

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Microbiological Research

j o ur na l ho m e p a g e :w w w . e l s e v i e r . d e / m i c r e s

Metal ion dependence of DNA cleavage by SepMI and EhoI restriction endonucleases

AbdelkarimBelkebir^∗,HoussineAzeddoug^∗

LaboratoiredeBiochimieetdeBiologieMoléculaire,FacultédesSciences,UniversitéHassanII-AinChock–Casablanca,km8,routed’ElJadidaBP.5366,Casablanca,Morocco

a r t i c l e i n f o

Articlehistory:

Received13January2012 Receivedinrevisedform4July2012 Accepted12August2012

Keywords:

Restrictionendonuclease Divalentmetalions Steadystatekinetics DNA-bindingproteins

a b s t r a c t

MostoftypeIIrestrictionendonucleasesshowanabsoluterequirementfordivalentmetalions as cofactorsforDNAcleavage.WhileMg²⁺isthenaturalcofactorothermetalionscansubstituteitand mediatethecatalysis,howeverCa²⁺(alone)onlysupportsDNAbinding.ToinvestigatetheroleofMg²⁺in DNAcleavagebyrestrictionendonucleases,wehavestudiedtheMg²⁺andMn²⁺concentrationdepend- enceofDNAcleavagebySepMIandEhoI.DigestionreactionswerecarriedoutatdifferentMg²⁺and Mn²⁺concentrationsatconstantionicstrength.Theseenzymesshoweddifferentbehaviorregardingthe ionsrequirement,SepMIreachednearmaximallevelofactivitybetween10and20mMwhilenoactiv- itywasdetectedinthepresenceofMn²⁺andinthepresenceofCa²⁺cleavageactivitywassignificantly decreased.However,EhoIwasmorehighlyactiveinthepresenceofMn²⁺thaninthepresenceofMg²⁺

andcanbeactivatedbyCa²⁺.Ourresultsproposethetwo-metalionmechanismforEhoIandtheone- metalionmechanismforSepMIrestrictionendonuclease.Theanalysisofthekineticparametersunder steadystateconditionsshowedthatSepMIhadaKmvalueforpTrcHisBDNAof6.15nMandaVmaxof 1.79×10⁻²nMmin⁻¹,whileEhoIhadaKmforpUC19plasmidof8.66nMandaVmaxof2×10⁻²nMmin⁻¹.

© 2012 Elsevier GmbH. All rights reserved.

1. Introduction

Divalentmetalionsarerequiredascofactorsformanyenzymes thatprocessnucleicacidsincludingDNAandRNApolymerases, ribozymes(SteitzandSteitz1993),nucleases(Suck1992),trans- posases, many recombination enzymes (Rice et al. 1996) and restrictionendonucleases(Pingoudetal.2009).Althoughmanyof theenzymesactingonnucleicacidsrequiredivalentcationslike Ca²⁺,Mn²⁺orZn²⁺(Gerlt1993;Wilcox1996),thenaturalcofactor forthemajorityoftheseenzymesisMg²⁺whileprocessingDNA, owingtothehighnegativechargedensityofDNAandrelatively tothehighnaturalabundanceofMg²⁺(MaguireandCowan2002;

Cowan1995).Invitro,theMg²⁺-dependentenzymesthatprocess nucleicacidscanoftenmediatethephosphoryl-transferreactions withMn²⁺insteadofMg²⁺.However,suchreplacementgenerally leadstoreducedefficiencyoftheenzymaticactivityandinmany casesMn²⁺altersthespecificityandtheactivityoftheenzyme.

The type II restriction endonucleases of the PD...D/EXK superfamily whichcleave4–8basepairdyad-symmetricrecog- nitionsequencesareexcellentmodelsforstudyingprotein–DNA

∗Correspondingauthors.

E-mailaddresses:[email protected](A.Belkebir), [email protected](H.Azeddoug).

interactionsdue totheirhighspecificitytowardtherecognition sequence.Theyshowanabsoluterequirementfordivalentmetal ions (Roberts and Halford 1993; Pingoud et al. 2009) and are uniqueamonghydrolyticenzymesintheirchallengetocatalyze in a charge repulsive, polyanionic context the cleavage of the phosphodiesterbond, whichis oneofthemoststablebondsin biochemistry(Williamset al.1999).Restrictionenzymesofthis superfamily show a commonstructural core consisting of four

␤-strandsandone␣-helix(Nivetal.2007;Scheuring-Vanamee andAggarwal2004).However,twofamiliescanbedistinguished (EcoRI-familyandEcoRV-family).TheEcoRI-familybindstheDNA fromthemajorgroovesideandproducescohesiveendswith5- overhangs,whereastheEcoRV-familyapproachestheDNAfrom theminorgroovesideandproducesbluntends.Thissuperfamily ischaracterizedbythePD...D/EXKmotif,whichwasproposedto represent thecatalytic centre and theMg²⁺ binding site. X-ray crystallographicanalysisofrestrictionsenzymesindifferentmetal- boundstateshaveelucidatedthecatalyticroleofMg²⁺andsplitthe typeIIrestrictionsendonucleasesintotwomaindifferentgroups withregardtotheirmetalbindingcapabilities.Thus,twocleavage mechanismswereproposedinwhichoneortwometalionsare involved(GalburtandStoddard2002).CrystalstructureofEcoRI and BglII-likeenzyme showedthat theycontaina single metal bindingsitepersubunit.Furthermore,co-crystalstructureofEcoRI- DNAinthepresenceMn²⁺(1QPS)showedthatasinglemetalion 0944-5013/$–seefrontmatter© 2012 Elsevier GmbH. All rights reserved.

http://dx.doi.org/10.1016/j.micres.2012.08.003

silephosphatefromthenucleophilicwatermoleculeandiswell positionedtoactivateawatermoleculeforprotonationoftheleav- inggroupoxygenandtohelp stabilizethenegativechargethat developsinthetransitionstate(Hortonetal.2004).

Inthispaper,wehaveinvestigatedthemetaliondependence andmetalioncooperativityinDNAphosphodiesterbondhydroly- sisbySepMIandEhoIrestrictionendonucleases.Cleavagereactions havebeen performedin thepresence of two differentdivalent metalions(Mg²⁺/Mn²⁺andCa²⁺)andtoelucidatetheroleofCa²⁺

inrestrictionactivity,DNAbindingexperimentswerealsocarried out.

2. Materialsandmethods

2.1. Bacterialstrainsandenzymespurification

AllchemicalswerereagentgradeandweremadeupinNanop- urewaterandpassedthrougha0.2␮mporesizefilter.SepMIwhich recognizesandcleavesGATATCwaspurifiedtoapparenthomo- geneityfromStaphylococcusepidermidisandEhoI(CTGCAG)from Enterobacterhormaecheibyathree-columnprocedureasdescribed previously (Belkebir and Azeddoug 2012). Protein concentra- tionsweredeterminedbyA₂₈₀ readings.Commercialrestriction enzymeswerefrom New England Biolabs.AmpliTaq Gold DNA polymerase was from Applied BioSystem (CA, USA). Different enzymequantitiesweretestedtodeterminearangeofproteins showinglessthan75%ofactivityinoptimalconditionsin20min.

Enzymeconcentrationwasreportedasunit/␮land1unitisdefined astheamountofenzymetocompletelyhydrolyze1␮goflambda DNAat37^◦Cin1hinavolumeof30␮l.

2.2. DNAsubstrates

pUC19andpTrcHisBDNAsubstratesformetalionsdependence experimentswerepreparedfromtransformedE.coliBL21(␭DE3).

Plasmidswereisolatedfrom100mlculturesusingtheSigmaGenE- lutePlasmidMiniprepkit.SubstratesforDNAbindingexperiments werepreparedbyPCRreactionofa250bplinearDNAfragment containingasinglecopyoftherecognitionsite,asdescribedear- lier(SambrookandRussell2001).TheconcentrationofDNAswas estimatedbymeasuringabsorptionat260nm.

2.3. MetaliondependenceofDNAcleavage

Divalentmetaliondependence assayofSepMIandEhoIwas performedinthepresenceofdifferentmetalionsatvariouscon- centrations. Cleavage reactions were carried out at 37^◦C in a bufferofconstant ionicstrengthcontaining10mMTris–HClpH 7.9,0.1mg/mlbovineserumalbumin,5mM2-mercaptoethanol, 0–50mMdivalentmetalions(MgCl₂orMnCl₂)and16nMofpUC19 (forEhoI)or30nMofpTrcHisB(forSepMI).NaClconcentrationwas

2mMEDTA)at700V/h.Gelswerestainedwithethidiumbromide (0.5␮g/ml)for15minandsubsequentlydestainedtwiceinwater for10min.DestainedgelswerephotographedusingaDOC-PRINT DP-001-FDCgeldocumentationsystem(VilberLoumate,France) andbandsintensitywasdeterminedusingImageQuantsoftware v5.0.Ascleavageofsupercoiledplasmids(SC)withonesinglesite occursinasequentialmanner,firstinonestrandtogivetheopen circle(OC)formoftheDNAthenintheotherstrand,togivethefull- lengthlinear(FLL)form,therelativeactivityrateswerecalculated fromtheincreaseintheamountofproduct(FLL),whichisplotted versusmetalionconcentration.Experimentswerecarriedoutat leasttwice.

ToassesstheeffectofCa²⁺onDNAcleavageinthepresenceof Mg²⁺,digestionreactionswerecarriedoutat37^◦Cfor20minina cleavagebufferofconstantionicstrengthcontaining0–5mMCa²⁺

inthepresenceofMg²⁺(0.5mM/5mM/10mM).

2.4. Steady-stateDNAkineticassays

Steady-state time course reactions under standard cleavage conditionswerecarriedoutat37^◦Cinanassaybuffercontaining 10mMTris–HClpH7.9,0.1mg/mlbovineserumalbumin,5mM 2-mercaptoethanol,10mMMgCl2,100mMNaCl,5–100nMDNA.

Aliquotsof20␮lwerewithdrawnat8differenttimepointsand thereactionswereterminated asdescribed above.The amount ofproductformedwasexpressedasmolarconcentrationandthe initial velocity of substrate cleavage wasdetermined fromthe linear regionof thereaction progress curveand normalizedto enzymeconcentration.Michaelis–Mentenconstants(KmandVmax) weredeterminedbyfittingthedoublereciprocal(1/Vversus1/[S]) Lineweaver–Burkplot(Segal1976).

2.5. Electrophoreticmobilityshiftassay

DNAbindingexperimentswereanalyzedbythegelmobility- shift assay, using a 250-bp DNA fragment containing a single SepMIorEhoIrecognitionsequence.Differentamountsofenzyme (0.2–3U)wereincubatedat25^◦Cwith50nMofDNAinthebind- ingbuffer(20mMTris–HClpH7.4,0.1mg/mlBSA,50mMNaCl, 5mM2-mercaptoethanoland5mMCaCl2),after30minaliquots of20␮lweremixedwith5␮lofthebindingbuffercontaining40%

(w/v)sucrose.Electrophoresiswascarriedouton10cm×10cm 6%polyacrylamidegel(29/1arcylamide/bisacrylamide)atroom temperaturein40mMTris–acetatepH7.9,2mMEDTAand5mM CaCl₂for16hat3V/cm.FractionofboundandunboundDNAwas quantifiedasdescribedabove.Thenormalizeddatawereplotted aspercentageoffractionboundversusproteinamountandcurve fittingwereperformedusingGraphPadPrism5software.

Fig.1. EffectofMg²⁺andMn²⁺concentrationonDNAcleavagebySepMIandEhoI.TherelativecleavageratesatdifferentconcentrationsofMg²⁺andMn²⁺arerepresented inblueandredrespectivelyforSepMI(a)andEhoI(b).Thehighestcleavageratemeasuredforeachenzymewassetto100%.(Forinterpretationofthereferencestocolour inthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)

3. Resultsanddiscussion

3.1. EffectofdivalentmetalionsonSepMIandEhoIactivity

PreviousstudieshaveestablishedthatmostoftypeIIrestric- tionendonucleasesexceptforBmrI,BfiIandPabIrequiredivalent metalionstosupportDNAbindingandcleavage(Baoetal.2008;

Miyazonoetal.2007;Sapranauskasetal.2000).Althoughthephys- iologicalmetal ionfor thebacterialenzymesappearstobethe magnesium,theycanutilizeavarietyofdivalentcationsforinvitro DNAcleavagereaction,thisincludesMn²⁺,Ca²⁺,Fe²⁺,Co²⁺,Ni²⁺, Zn²⁺orCd²⁺,dependingontheenzyme(PingoudandJeltsch1997, 2001;Cowan2004).ToinvestigatetherequirementsofSepMIand EhoIwehaveincubatedtheenzymeswithdifferentmetalioncon- centrationsrangingfrom0to50mMinthepresenceofplasmid DNA containinga singlecopy of therecognitionsequence as a substrateandthenanalyzedtheproductsbygelelectrophoresis.

Toavoidtheeffects ofnonspecificshielding ofDNA bindingon theionconcentrationdependence ofDNAcleavagebythevari- ousconcentrationsofdivalentmetalions,constantionicstrength hasbeenadjustedineachreactionto150mM.Theresultsdemon- stratedthatforbothrestrictionendonucleases,cleavageactivity showedanabsoluterequirementfordivalentmetalions.Themax- imumlevelofactivitywasseenforSepMIinthepresenceofMg²⁺

atconcentrationsbetween10and20mM,however nocleavage activitywasobservedinthepresenceofMn²⁺orCa²⁺.Thusonly Mg²⁺actsefficientlyasacofactorFig.1ashowstheeffectsofmetal ionconcentrationonSepMI-mediatedDNAcleavage.ForEhoI,near maximallevelofplasmidhydrolysishasbeenobservedinthepres- enceofMg²⁺atconcentrationsaround5mM.ContrarilytoSepMI, EhoIwasalsoactiveinthepresenceofMn²⁺withahighercleav- ageratenearly2.5-foldthanthatofMg²⁺.Thetitrationcurvefor Mn²⁺showedasharpincreaseinactivitybetween0.05and0.1mM whileconcentrationsabove1mMcauseinhibitionoftheenzyme.

TheapparentaffinityofEhoIforMn²⁺ishigherthanthatforMg²⁺

asestimatedonthebasisofthecleavageactivityversusconcen- trationprofiles(Fig.1b).Thus,thedivalentcationsMg²⁺orMn²⁺

arecapitalcofactorsforDNAhydrolysisbytheseenzymes,how- everathigherconcentrationstheyinhibittherestrictionactivity.

Thisinhibitioncanbeattributedspecificallytotheeffectofeach metalion,sinceinhibitionoccurredatdifferentmetalionconcen- tration(>5mMMg²⁺or>1mMMn²⁺forEhoIand>20mMMg²⁺

forSepMI)inabufferofconstantionicstrength.Thedifferencein theeffectbetweenMg²⁺andMn²⁺oncleavageratecanbedueto thedissimilarityinthebindingofthesedivalentmetalionstothe

activesite,orbythenon-interventionofsomemetalionsinthe catalysis,asreportedforsomerestrictionendonucleases(Horton etal.1998;Nastrietal.1997).

TherestrictionendonucleaseEcoRValsoshowednocleavage activityintheabsenceofdivalentmetalions,howeveritcanbind toallDNAsequenceswithequalaffinity(Engleretal.1997).The naturalcofactorMg²⁺canbesubstitutedbyotherdivalentionslike Co²⁺orMn²⁺,howeverthisreplacementresultsinachangeinthe cleavagerate.Theturnover ratewithCo²⁺is aboutthreetimes higherthanthatwithMg²⁺,whileMn²⁺yieldeda cleavagerate tentimesslower(Baldwinetal.1999;Vipondetal.1995).Thus, divalentmetalionsplayacentralroleinDNAcleavagebytypeII restrictionendonucleases.Therestrictionenzymesthatbelongto thePD...D/EXK family,canuseotherdivalentmetalionsinstead ofMg²⁺tosupportcleavage(inparticularMn²⁺orFe²⁺,Co²⁺,Ni²⁺, Zn²⁺,Cd²⁺,dependingontheenzyme),howeverCa²⁺isnotaneffec- tivecofactor(Pingoudetal.2005).Restrictionendonucleasesof otherfamiliesarealsodependentondivalentmetalions,theycan usedifferentdivalentcationsincludingCa²⁺(Kriukiene2006).For exampleKpnIamemberoftheHNHfamilyandCfr42Iamember oftheGIYYIGfamily,Mg²⁺canbereplacedbyCa²⁺asacofactorfor cleavageactivity(Gasiunasetal.2008;Saravananetal.2007).

3.2. EffectofCa²⁺onMg²⁺-mediatedDNAcleavage

TheeffectofCa²⁺onMg²⁺-dependentDNAhydrolysisbySepMI and EhoI endonucleases were investigated by measuring DNA cleavagerateswithacombinationofMg²⁺andCa²⁺.Thecleav- agereactionswerecarriedoutatdifferentconcentrationsofMg²⁺

(0.5mM,5mMand10mM)inaconstantionicstrengthbuffer.The resultsofourDNAcleavageexperimentsshowedforbothenzymes nodetectablecleavageactivityinthepresenceofCa²⁺alone(data notshown).AsobservedinFig.2aCa²⁺metalionscausedasignif- icantdecreaseinthecleavagerateofSepMIactivityascompared withdigestioninthepresenceofMg²⁺alone.DNAcleavagerates weredecreasedastheconcentrationsofCa²⁺wereincreasednearly 3.5-foldlowerinthepresenceof10mMMg²⁺,11-foldlowerinthe presenceof5mMMg²⁺at5mMofCa²⁺,whilethisconcentration ofCa²⁺resultsinnodetectablecleavageactivityinthepresenceof 0.5mMMg²⁺.However,theMg²⁺-dependentDNAcleavageactiv- ityofEhoIwasstimulatedbyCa²⁺inthemetal-ionconcentration rangetested.EhoIactivitywithMg²⁺showedabiphasicresponse toCa²⁺ metalions:stimulation atlow concentrationsgenerally around10–25-foldexcessofMg²⁺overCa²⁺andinhibitionathigh concentrations.ThestimulatoryeffectofCa²⁺appearstobemore

Fig.2. TheeffectofCa²⁺onMg²⁺-mediatedDNAcleavagebySepMIandEhoI.ThegraybarsrepresentthecleavageratebySepMIorEhoI,inthepresenceofdifferentCa²⁺

concentrationatagivenMg²⁺concentration(0.5mM,5mMand10mM).TheblackbarsrepresentthecleavageratewithoutCa²⁺atthegivenMg²⁺concentration.

dependentontheMg²⁺/Ca²⁺rationthanontheCa²⁺concentration.

TheMg²⁺-mediatedDNAcleavageratebyEhoIwasincreasedby about1.5-foldattheMg²⁺concentrationstested.Thisresultimplies thatin thepresenceof DNAsubstrate andunder thedescribed experimentalconditions,EhoIhasaCa²⁺bindingsitewithanappar- entaffinity lessthan5mM (Fig.2b).Theseresultsindicatethat SepMIrequiresonlyonemetalionperactivesiteandisinhibited whenMg²⁺issubstitutedbyCa²⁺.Thus,weproposeaone-metal mechanismfortheDNAphosphodiesterbandhydrolysisbySepMI.

HoweverEhoIrequirestwometalionsandthesubstitutionofone byCa²⁺stimulatesthecleavageactivity.Thefirstsite(siteA)shows ahigherapparentaffinityforMn²⁺thanthatforMg²⁺,inthissite (catalytic)Mg²⁺orMn²⁺isrequiredforspecificbindingandcleav- age.Thesecondsite(siteB:modulator)isinhibitingifoccupiedby anMg²⁺orMn²⁺metalionandisactivatingifaCa²⁺metalionis bound(Vipondetal.1995;Beerninketal.2001).

LikeEhoItheactivityofEcoRVwithMg²⁺isstimulatedinthe presenceoflowCa²⁺concentrationshoweverconcentrationsmore than2mM ofCa²⁺ inhibitedtheenzyme activity(Vipondet al.

1995). Recentstudy of restrictionenzymesof theEcoRI family (BamHI,EcoRI,MboI,NgoMIV,PspGIandSsoII)showedthatthese enzymesarealsostimulatedbyCa²⁺toavariabledegreeandinhibi- tionalsooccurredabovecertainCa²⁺concentrationdependingon theenzymeandtheconcentrationsofMg²⁺(Pingoudetal.2009).

Unliketheseenzymes,thecleavageactivityofT5flapendonucle- aseandPvuIIrestrictionenzymewereinhibitedbytheaddition ofCa²⁺metalionsinthepresenceof thenaturalcofactorMg²⁺

(Sysonetal.2008;Prasannanetal.2010).X-raycrystallographic analysisofEcoRVproposedatwo-metalionmechanisminwhich theionboundat the90/74site facilitatestheformation ofthe nucleophilichydroxide,thissitecorrespondstothesiteA,while thesecondmetal,boundatthe74/45sitewhichcorrespondsto siteBisresponsibleforbothpositioningandactivatingtheattack- ingwatermolecule.ThemetalioninthesiteBhelpstostabilizethe transitionstateofthenucleophilicattackstep(Vipondetal.1995).

Moleculardynamic simulation publishedpreviously showed thatthemetalioninthesiteBleadstoastructuralchangeinthe DNAthatinducestoremovalofmostoftheroll,thuskeepingDNA intheoptimalconformationforcleavage.Moreover,itisobserved

thatEcoRVismoretightlyboundtoitsrecognitionsequenceinthe presenceofbothmetalions(Zahranetal.2011).Singleturnover experimentsofPvuIIamemberoftheEcoRVfamilyshowedthat thecleavageactivitycanbesupportedbyoneMg²⁺ionperactive site,howeverthetwometal-ionmechanismismoreeffectiveunder higherMg²⁺concentrations(XieandDupureur2009).Kineticand computationalstudiescarriedoutonrestrictionenzymesofthe EcoRIfamilyshowedthatDNAbindingandcleavagearesupported byonemetalion,whilethesecondmetalionplaysamodulatory role;itdecreasestheactivityathighionconcentrationorincrease activityinthecaseofCa²⁺(Pingoudetal.2009).

3.3. Steady-stateDNAkineticassays

TodeterminethekineticparametersofSepMIandEhoIunder steady-statereactionconditions,cleavagereactionswerecarried outwithpTrcHisBandpUC19DNArespectivelyatdifferentcon- centrationsand under saturating conditions of Mg²⁺ metal ion (10mM).Likeotherrestrictionendonucleases,SepMIandEhoIcat- alyzethecleavageofsupercoiledplasmids(SC)withonesinglesite inasequentialmanner.TheplasmidDNAiscleavedinitiallyinone strandtogivetheopencircle(OC)formoftheDNAtheninthesec- ondstrand,togivethefull-lengthlinear(FLL)form.Thecleavageof bothstrandsisoftenfasterthanthedissociationofenzymefromthe DNA(Gormleyetal.2000).Thefractionofthefull-lengthlinearDNA producedateachtime-pointwasusedtomeasuretheactivityofthe enzymes.Undertheconditionsstatedabove,thehydrolysisofall theplasmidDNAsrequiresmultiple-turnovercleavagereactions, eachinvolvesbindingofenzymetoaDNA molecule,catalyzing thecleavagethendissociatingfromtheproductbeforeactingupon anotherDNAmolecule.DNAcleavagebySepMIandEhoIrestric- tionendonucleasesdisplayedbiphasickinetics,arapidburstphase followedbyaslowersteady-stateproductrelease(datanotshown) whichisastrongevidenceforamultistepmechanismconsistingof afastfirststepandaslowersecondstep.Thispatternwasobserved atallconcentrationsofDNA(Fig.3a).Thesedataimplythatthe releaseofDNAproductfromtheenzymecouldbetheslowerstep inthisreactionandtheenzymesfunctionscatalyticallyratherthan stoichiometrically. The initialvelocity of DNA cleavage at each