TopoisomeraseIIDrugScreeningKitProductDescription
TopoIIinhibitorshavebeenshowntopossesseffectiveantibacterial,antiviralandanticanceractivity.IdentificationoftopoIIinhibitorsandantagoNISTsrequirestractablebiochemicalassaysemployingpurifiedenzyme. TheTopoisomeraseIIDrugScreeningKitcontainsthereagentsnecessarytoscreenforagentsthataffectenzymaticactivity. Toallowthecustomermaximumflexibility,purifiedtopoisomeraseIImaybepurchasedseparatelyfromthekit(TG2000H). Thiskitwillallowdetectionoftwoclassesoftopoisomeraseinhibitors:thosethatstimulateformationofcleavablecomplexesandthosethatantagonizetopoIIactionontheDNA. Alsoincludedinthiskitisdetaileddocumentationondesignandinterpretationofinhibitiondata. Knowninhibitorsareincludedascontrols. TheDNAsubstrate(pHOT1)includedinthisassayisidealforthesestudiesbecauseitcontainsasingle,highaffinitytopoIIcleavagesite(notethattopoIIisnotparticularlysequencespecific).TheassaysystemisbaseduponevaluatingtheformationofDNAcleavageproductswhichmaybenicked,opencircularDNAorlinearDNA.Theproductsarethenresolvedbyethidiumbromidegelelectrophoresisasdescribedinthekit.
TopoisomeraseIIDrugScreeningKitContents(representativeof100assaykitsize):
-SupercoiledDNA[pHOT1DNA]25ug
-MarkersarelinearpHOT1DNAandopencircularDNA
-10XTopoisomeraseIIassaybuffercomponents*
*ToimprovestABIlity,the10xTopoIIAssaybufferisprovidedastwocomponents: BufferA(noATP)andBufferB(ATP).TheComplete10xAssaybufferismadefresheachtimebycombiningBufferAandB.
-Sodiumdodecylsulfate(10%)
-10XGelloADIngbuffer(bromophenolblue,glycerol)
-Controlinhibitor,Etoposide
-ProteinaseK
-Detailedinstructionmanual
Note:TopoIIenzymeissoldseparately.
TopoIIDrugScreeningBackground:
ThisisacleavagebaseddetectionkitthatwillrevealwhetheraparticularagenteitherantagonizestheactionoftopoIIoraffectsthecleavage/religationcycle. Reactionsareassembledinmicrocentrifugetubesusingreagentssuppliedinthekit(buffer,DNA,controlinhibitor). ThiskitisdesignedtoworkwithpurifiedtopoisomeraseIIprovidedbyTopoGEN. Afteraddingenzyme,thereactionsareincubated15-30min(37°C),andterminatedbyadditionofSDS.
Atypicalreaction:
- H2O:Volumevariable,makeuptovolume(20ulinthiscase)
- 10XAssaybuffer2µl(madefreshfromBuffersAandB,seeKitdetails)
- DNA(supercoiled)1µl
- TestCompoundorcontrolinhibitor:Variable(limitedbysolventeffectsasaddressedincontrols)
- **PurifiedTopoisomeraseII:4-6Units(enzymesoldseparately)
- Incubate15-30minutesat37°C
- Add2µlSDS;digestwithproteinaseK(50ug/ml)
- Add0.1volsampleloadingdye(supplied)
- Loadgel:1%agarosecontaining0.5ug/mlethidiumbromidealongwiththemarkerDNAsincludedinthekit.
ThereactionsindicatedaboveeachlaneinthefigurewerecarriedoutusingtheenzymeprovidedbyTopoGENand1ulofDNA(0.25ugofformIpHOT-1,asprovidedineachkit). Thereactionproductswereanalyzedona1%agarosegel;therunningbuffercontained0.5ug/mlethidiumbromide. Thegelwasthendestainedindistilledwaterpriortophotodocumentation. Formationofthecleavablecomplex(linearDNAspecies)isclearlyvisIBLe. Additionally,relaxationofsupercoiledDNAsubstrate(formIpRYGDNA)wasinhibitedinallreactionsthatcontainedVM26. ThekitcontainsmarkerDNAs,VM-26controlinhibitor,allbuffersandcompleteinstructionsonusingthereagentstoscreenforinhibitorsoftopoisomeraseII. ThemanualspecifiesconditionsforgelelectrophoresistoresolveallDNAforms;publicationqualitygelswillresult.
**CleavagereactionsrequiremoretopoIIthanrelaxationassayssincetheformerisastoichiometricreactionofenzyme. Forexample,theabovereactionscontained4unitsofenzyme(unitsdefinedbydecatenationactivity).
ReviewsandCitations:
- Sudan,S.,&Rupasinghe,H.V.(2014).Flavonoid-EnrichedAppleFractionAF4InducesCellCycleArrest,DNATopoisomeraseIIInhibition,andApoptosisinHumanLiverCancerHepG2Cells.Nutritionandcancer,ahead-of-print(2014):1-10. doi:10.1080/01635581.2014.951733
- ChengMH,YangYC,WongYH,ChenTR,LeeCY,YangCC,ChenSH,YangIN,YangYS,HuangHS,YangCY,HuangMS,ChiuHF:B1,anoveltopoisomeraseIIinhibitor,inducesapoptosisandcellcycleG1arrestinlungadenocarcinomaA549cells. Anti-CancerDrugs2012,23(2):191-199. doi:10.1097/CAD.0b013e32834cd277
- ShiW,MarcusSL,LowaryTL:CytotoxicityandtopoisomeraseI/IIinhibitionofglycosylated2-phenyl-indoles,2-phenyl-benzo[b]thiophenesand2-phenyl-benzy[b]furans. BioorganicandMedicinalChemistry2010. doi:10.1016/j.bmc.2010.10.054
- GongY,FirestoneGL,BjeldanesLF:3,3'-DiindolylmethaneisanoveltopoisomeraseIIacatalyticinhibitorthatinducesS-phaseretardationandmitoticdelayinhumanhepatomaHepG2cells. MolecularPharmacology2006,69(4):1320-1327.
