TopoGEN/Kinetoplast DNA (kDNA)/TG20131/25ug

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.