TopoisomeraseIVDrugScreeningKitBackground

TheTopoisomeraseIVDrugScreeningKitcontainsreagentsnecessarytoidentifycompoundsthataffectenzymeactivity. NotethatthiskitdoesnotincludeE.colitopoisomeraseIVenyzme,whichissoldseparately. Thiskitwillallowdetectionoftwokindsoftopoisomeraseinhibitors:thosethatstimulateformationofcleavablecomplexesandthosethatantagonizetopoisomerase(topo)IVactionontheDNA. SincetopoIVisatypeIIenzyme,wemayrefertoitaseithertopoIVortopoII. AknowntopoIIpoisonisincludedasacontrol. Theassaysystemisbaseduponincubatingsupercoiledplasmid(pHOT1)withTopoIVandevaluatingtheformationofDNAcleavageproductswhichmaybenicked,opencircularDNAorlinearDNA. Theproductsarethenresolvedbyethidiumbromidegelelectrophoresisasdescribedinthekit.

TopoisomeraseIVDrugScreeningKitContents(suitablefor100assays):

-pHOT1DNA,supercoiled25ug
-MarkerDNA,LinearpHOT1
-IncompleteBufferAcontainsthefollowing:200mMHEPES-KOH[pH7.6],500mMpotassiumglutamate,50mMMagnesiumAcetate,50mMDithiothreitol,250ugBSA/ml(500ul)*
-ATPBufferBcontains100mMATP*

*Thesetwobuffersmustbemixedbeforeusetomakethefinal5xCompleteTopoIVAssayBuffer(A+B)asfollows:add0.1volumeofBufferBto1.0volumeBufferA.PrepareonlyasmuchoftheCompleteTopoIVAssayBuffer(A+B)asneededforeachdayorsetofexperiments.Notethatthisbuffershouldbekeptoniceandusedthesameday,thendiscarded.

-Ciprofloxacincontroldrug
-10%sodiumdodecylsulfate(SDS)
-10xgelloADIngbuffer
-ProteinaseK
-Detailedinstructionmanual

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.

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