S.aureusDNAGyraseProductBackground
HighlypurifiedS.aureusDNAgyraseisatypeIItopoisomeraseencodedbytwogenesGyrAandGyrBfromstaphylococcusaureus. DNAgyraseisanessentialtopoisomerasethatsupercoilsDNAthroughaprocessofstrandbreakage/resealingandDNAwrapping(3). AsatypeIIenzyme,gyraseisuniqueinitsABIlitytonegativelysupercoilarelaxedplasmidDNAsubstrate. S.aureusDNAgyraseisalsothetargetforquinolone-basedantibacterialagentswhichactbysubvertingtheenzymeintoaDNAdamagingagent. TopoGENofferspurifiedDNAgyrasefromthegrampositivebacterium,Staphylococcusaureusforuseinallaspectsofdrugdevelopmentandscreeningassays. TheenzymeisveryactiveindecatenatingandsupercoilingKDNA(TG2013)butwillalsonegativelysupercoilrelaxed plasmidDNA(TG2037). TheS.aureusenzymeisaheterotetramer ofGyrA2GyrB2andispurifiedasHistaggedsubunitsthatarereassembledtomakeactiveenzyme(4).

S.aureusDNAGyraseQualityControlTests
DNAgyrasesubunitswereclonedoverexpressedandpurifiedusingtheproprietarycompanymethods. AsinglebandonSDS-PAGEwasdetectedbyCBstainingforeachsubunit. CrosscontaminationbytopoIwasassessedbyassayingforrelaxationofsupercoiledDNAunderconditionsoptimizedfortypeIactivity. Undertheseconditions,after2hoursofincubationwithsupercoiledplasmidDNA,norelaxationproductsweredetectable.

AtestfornucleasecontaminationwascarriedoutbyassayingfortheformationoflinearKDNAandlinearplasmidDNA.  Incubationsof1µgofcatenatedKDNAorsupercoiledDNA(4hrs.at37°Cinthepresenceof10mMMgCl2)wereperformed. LinearDNAorbreakdownproductswerenotgeneratedundertheseconditions.

Thesubunitsarebetterthan95%purebaseduponSDS-PAGE.Neithersubunitisactiveintheabsenceoftheotherandneithersubunitdisplaysnucleolyticactivity. Thesedatashowthathost(E.coli)isnotcontributingtotheactivityofindividualsubunitsof S.aureusgyrAorB. ThiswasconfirmedbyWesternblottingprobingswithanti-GyrAIgGspecifictoE.coli(datanotshown).

AssayConditions
TopoGENprovidessamplesof*threebuffersthatareusedtoassayS.aureusDNAGyrase. Thebuffersare:
5XAssayStock(0.5mlprovided)containsthefollowingsolutes:

• 375mMTris-HCl(pH7.5)
• 37.5mMMgCl2
• 37.5mMDTT
• 0.375mg/mlBSA
• 150mMKCl
• 10XATPStock: 20mMATP(0.25mL). Enzymerequires2.0mMATPFINALCONCENTRATION
• 5XPotassiumGlutamateStock: 2.5M(0.25mL). Enzymerequries500mMK-GluFINALCONCENTRATION.

*Note:ThesebuffersareprovidedtoallowthecustomertoestablishaworkingcontrolfortheGyraseassay. Additionalbuffermayberequired. Storeallbuffersat-20°C.

Assaysshouldbeperformedinafinalvolumeof20ulbyaddingappropriateamountsofbuffers,DNAenzyme(addenzymelasttoinitiatetheassay). Typicalassaywouldbe:

(Enzymedilutedtoworkingconcentrationusingdilutionbufferortitrateas2foldserialdilutions.)
Incubate30min37°C,stopbyadditionofSDSto1%(notincluded);addBromophenolblue/glycerolloADIngdye,run1%agarosegelwithrelaxed/supercoiledMarkers. Optional: digestwithproteinaseKandrepurifyDNAbyphenolextraction. GelsshouldbestainedwithEthidiumBromide(0.5ug/ml20-30min),destainedinwaterfor15-30minatroomtemperatureandphotodocumented.

DilutionBuffer
Ifnecessary,dilutetheenzymeusingthefollowingbuffer(1x)suppliedwiththeenzyme.
50mMTris-HCl[pH7.5],100mMNaCl,2mMb-DTT,1mMEDTA,10%glycerol.

ModificationsoftheGyraseAssay: DetectingDNAcleavages.
GyraseisatargetforseveralantibioticsthatinducetheenzymetocleavetheplasmidDNAsubstrate(3,5).  Quinolonedrugsareknowntoinducecleavagesconcurrentwithcovalentcomplexformation;thus,todetectsuchcomplexesindrugscreeningexperiments,itisessentialthatproteinaseKdigestionsbecarriedoutandthatreactionproductsrepurifiedbyphenol/chloroformextraction. Additionally,theconditionsthatareoptimalforcleavagedetection,differslightlyfromthoseoptimizedforcatalyticassays. ForDNAGyrase,wefindthatreducedpotassiumglutamate(200mMfinalconcentration)isidealforcleavagecomplexformation. 

ToresolvenickedandlinearDNAcleavages,werecommendrunning1%agarosegelscontaining0.5u/mlEBinthegelandrunningbuffer. Destainfor15minpriortophotodocumentingtheresults. ItisessentialtoincludelinearDNAmarkersinthesegels.

• Distilledwater-8ul
• 5XAssaystock-4ul
• 10XATPStock-2ul
• 5XK-GluStock-4ul
• 0.1ugsupercoiledPlasmidDNA-1ul
• Purifiedenzyme(variable)-1ulAddedlast
  • The1xcleavagebuffercontains:
  • 75mMTris-HCl(pH7.5)
  • 7.5mMMgCl2
  • 7.5mMDTT
  • 2mMATP
  • 75ug/mlBSA
  • 30mMKCl
  • 200mMK-Glu

ReviewsandCitations
PhillipsJW,GoetzMA,SmithSK,ZinkDL,PolishookJ,OnishiR,SaloweS,WiltsieJ,AlloccoJ,SigmundJ,DorsoK,LeeS,SkwishS,delaCruzM,MartinJ,VicenteF,GenilloudO,LuJ,PainterRE,YoungK,OverbyeK,DonaldRGK,SinghSB:DiscoveryofKiBDelomycin,APotentNewClassofBacterialTypeIITopoisomeraseInhibitorbyChemical-GeneticProfilinginStaphylococcusaureus. CellChemistryandBIOLOGy2011,18:955-965. doi:10.1016/j.chembiol.2011.06.011.

PengH,MariansK:DNATopoisomeraseProtocolsVolumeI1999.p.163TGVi

WangJ:DNATopoisomerases. Ann.Rev.Biochem1996,65:635-692

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