HumanTopoisomeraseIIAssayKitProductDescription

ThiskitisdesignedtoallowquickandspecificassaysforeukaryotictypeIIDNAtopoisomerases.ThiskitfacilitatesthepurificationandcharacterizationoftypeIIenzymesandcontainsallreagentsnecessaryforroutineassaysoftypeIItopoisomerases.TheassayisbasedupondecatentationofkinetoplastDNA(kDNA)andbecauseitisspecificfortypeIIactivity(nottypeI),itcanbecarriedoutwithcrudecellextracts.ReactionproductsareresolvedusinganovelgelsystemdevelopedbyTopoGENthatallowsextremelyrapidandunambiguousdetectionoftopoisomeraseIIactivity.Theappropriatebuffers,DNAsubstratesandDNAMarkersareincluded.PurifiedtopoIIisnotincludedinthiskit(TG2000H);however,topoisomeraseIIdecatenatedandlinearDNAmarkersareincludedtoallowclearandfacileidentificationoftopoIIactivityintheextractsorfractionsdefinedbytheuser.Theassaykitcanalsobeusedinconjunctionwithpurifiedenzyme(suppliedbyTopoGEN)todetectinhibitorsoftopoII.ThekDNAassaywilldetectpoisonsthatstimulateDNAcleavagebytopoIIaswellasagentsthatsimplyinhibitcatalyticactivity.TheadvantageofkDNAbasedassaysisthattheassaysarefastandeasytoallowactivityguidedpurificationofinhibitorsfromcrudemixturesorextracts.

HumanTopoisomeraseIIAssayKitContents(For100assaykitsize):

-KinetoplastDNA(kDNA)20ug
-MarkerlinearkDNAingelloADIngbuffer
-MarkerdecatenatedkDNA(topoIItreated)
-10XTopoisomeraseIIassaybuffer*
-5XStopbuffer/gelloadingdye
-Detailedinstructionmanual

*NEW:ToimprovestABIlity,the10xTopoIIAssaybufferisprovidedastwocomponents:BufferA(noATP)andBufferB(ATP).TheComplete10xAssaybufferismadefresheachtimebycombiningBufferAandB.

TitrationofTopoIIActivityUsingtheHumanTopoisomeraseIIAssayKit

Lane1:   200nglinearizedkDNA
Lane2:   200ngdecatenatedkDNA
Lane3:   NoTopoII
Lane4:   1:2dilutedTopoII
Lane5:   1:4dilutedTopoII
Lane6:   1:8dilutedTopoII
Lane7:   1:16dilutedTopoII
Lane8:   1:32dilutedTopoII
Lane9:   1:64dilutedTopoII

HumanTopoisomeraseIIEnzyme(TG2000H,soldseparately)wasincubatedwithkDNAfor15minat37°Cusingtheassaybuffersuppliedwiththekit.Theenzymeconcentrationwasapproximately2unitsperul. Reactionswereterminatedusingstopbufferandloadeddirectlyontoa1%agarosegelcontainingethidiumbromide(0.5ug/ml). Afterelectrophoresis,thegelwasdestainedfor30minandphotographed. Notethatthedecatenatedproductscontainopencircular(upperband)andcovalentlyclosedcircular(relaxed)minicircleDNA. NotealsothatlinearDNAmigratesbetweenthenickedandrelaxedspecies. ThenickedminicircularDNAispresentinallkDNApreparations. Additionally,theamountofnickedDNAmayvarybetweenKDNApreparation. LinearanddecatenatedkDNAmarkersaresuppliedinthekit. Itisimportanttorunthesemarkerstoidentifypositionsofdifferentforms. Whenassayingcrudeextracts,itisalsoimportanttorealizethatextractsoftenmaycontainUVfluorescingcontaminants(RNAorDNAbreakdownproductsforexample). Themarkerswillhelpinidentifyingsuchartifacts;however,weadvisethatyoualsorunonelaneofproteinextractwithoutkDNAsubstratetorevealthesecontaminants.

Complications:
Themostseriouscomplicationsarisewhenthereareinterferingproteinsorsubstancesintheextractbeingassayed. Crude,cellfreeextractsmaycontainexcessiveamountsofDNAbindingproteinsorpositivelychargedproteinsthatsticktotheDNAandinhibitenzymeaccess. Also,nucleasecontaminantsmaydegradeornickthekDNAsubstrateandthereforeobscuretheresults. Agoodwaytodealwiththisproblemincludescleaningupcrudeextractsbyammoniumsulfateprecipitationfollowedbycolumnchromatography. Also,bydilutingextractsand/oraddingatRNAcarrier(tocompetebasicproteins),onecansometimesminimizesuchproblems.

ReviewsandCitations:

• GardnerL,MalikR,ShimizuY,MullinsN,ElshamyWM:GemininoverexpressionpreventsthecompletionoftopoisomeraseIIαchromosomedecatenation,leadingtoaneuploidyinhumanmammaryepithelialcells. BreastCancerResearch2011,13(3):R53. doi:10.1186/bcr2884
• BowerJJ,KaracaGF,ZhouY,SimpsonDA,Cordeiro-StoneM,KaufmanWK:TopoisomeraseIIamaintainsgenomicstabilitythroughdecatenationG2checkpointsignaling. Oncogene2010,29(34):4787-4799. doi:10.1038/onc.2010.232
• Polycarpou-SchwarzM,MüllerK,DengerS,RiddellA,LewisJ,GannonF,ReidG:Thanatop:ANovel5-Nitrofuranthatisahighlyactive,cell-permeableinhibitoroftopoisomeraseII. CancerResearch2007,67:4451-4458. doi:10.1158/0008-5472.CAN-07-0393