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
