kDNAProductDescription
kDNAistheidealsubstratefortopoisomeraseIIassaysbecauseitisspecificfortypeIIreactionmechanisms. ResearcherscanevenassayforatypeIIenzymeinthepresenceoflargeexcessoftopoisomeraseI. Thus,kDNAworkswelltoquantifytypeIIactivityincrudecellextracts,whicharefrequentlyoverloadedwithtopoisomeraseI. ThekDNAsubstrateworksexceptionallywellwithhumantopoisomeraseIIaandIIbaswellasprokaryoticenzymes,suchasDNAgyraseortopoisomeraseIV. ThissubstrateispackagedwithourTopoIIAssayKit,TopoIIDrugKit,andGyraseAssayKits.
AssaysemployingcrudeextractsfortopoIIactivitybaseduponrelaxationofsupercoiledDNAcanbecomplicatedduetothepresenceoftopoIinpartiallypurifiedfractions. Additionalcomplicationsarisewithcontaminatingnucleaseactivity(duetoMg++)whichdegradeornickthesupercoiledsubstrate. TheseproblemscanbeavoidedbyusingacatenatedDNAsubstratepreparedfromthekinetoplastoftheinsecttrypanosomeCrithidiafasciculata. KinetoplastDNA(kDNA)isanaggregateofinterlockedDNAminicircles(mostly2.5kb)thatformextremelylargenetworksofhighmolecularweight. Asaresult,thesenetworksfailtoenteranagarosegel.UponincubationwithtopoII,whichengagesDNAinadoublestrandedbreakingandreunioncycle,minicircularDNAsareeffectivelyreleased(decatenated). Thedecatenatedminicirclesmoverapidlyintothegelowingtotheirsmallsize. ThisreactionwillnotoccurwithtopoisomeraseI.Theproductsofthereactioncanvaryasfollows:
QualityControlTests:
- ForcatenatedKDNAsubstrate,atleast90%oftheDNAwillberetainedinthewellofa1%agarosegel.
- DecatenationofeachbatchofKinetoplastDNAistestedwithpurifiedtopoisomeraseII.
StorageBuffer:
kDNAisstoredin10mMTris-Cl(pH7.5),and1mMEDTAattheconcentrationspecifiedwiththeproduct.
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.
