SDS-K+PrecipitationKit(invivo)ProductDescription

Thiskitcontainsreagentstotrapanddetectcovalenttopoisomerase/DNAintermediatesthatforminvivo(forexampleinsidecellsornucleiwithendogenoustopoisomerase).

KitContents:

-Camptothecin(10mM)
-Etoposide(10mM)
-10XTKEwashbuffer
-2.5MKCl
-CellularLysisSolution
-ADetailedManualfortheSDS-K+Precipitationalongwithcontrols

ThisTopoGENkitisdesignedtoquantifyformationoftheendogenouscovalentprotein/DNAintermediatesthatformwithgenomicDNA. DetectingthecomplexisgreatlyenhancedbytopopoisonsthatstABIlizethereaction(covalent)intermediate. SincethegenomicDNAisrADIoactivelytagged([3H]-TdR),theprocedureisquantitativeandcanbeusedtocompareoridentifydifferentinhibitorsinsitu. Radioactivityisnotsupplied;however,detailedinstructionsareincludedwhichdescribetheprocedure(forusewithcelllines).

ThefollowingmaterialsandreagentsareNOTsuppliedwiththiskitbutwillberequired:

95%and70%Ethanol
GlassFiberFilters(usedforTCAprecipitations,suchasWhatmanGF/C)
FiltrationmanifoldorsingleMilliporeFilterstack
alpha-[3H]-Thymidine
Scintillationvials,Scintillationfluidandcounter

Background:
BothtypeIandIItopoisomerasescanbetrappedinacovalentcomplexwithDNAbyaddingproteindenaturants(alkali,acidordetergents)whiletheenzymeisactivelyengagedinbreakingandresealingstepsonDNA. Althoughthedetailedmechanismoftrappingbydenaturationisnotknown,itisgenerallyacceptedthatcapturingthecovalentintermediateorcleavablecomplexisdifficultbecausetheintermediatehasarelativelyshortlifetime. Thus,withnormalamountsoftopoIorII(i.e.,sufficientenzymetoobservecatalysis),detectingthecovalentcomplexbydenaturationrequiresthepresenceoftopoinhibitorswhichtendtoextendthelifetimeofthecleavagecomplex. (Strictlyspeaking,thesetypesofinhibitorsareinfactbetterdescribedaspoisons.) Formationofacovalentintermediatecanbeexploitedasameansofidentifyingnoveltopopoisonsincethecomplexisafunctionalintermediateofthecatalyticcycleoftopoisomerase. Covalenttopo/DNAcomplexesaregeneratedbyaddingSDStoareactioninwhichtheenzymeisnickingandresealingDNA;afraction(typically<5%) of the topo is trapped in a covalent complex with DNA.  SDS binds only protein (not DNA) and non-covalently bound topo is irreversIBLyinactivated.UponadditionofKCl,insolublecrystalsofpotassiumdodecylsulfateform(werefertothisasSDS-K+)whichco-precipitatefreeproteinandonlythoseDNAscovalentlyboundtotopoI.IftheDNAislabeled,theamountoflabelintheprecipitateisameasureofDNAmoleculeswithcovalentlybound(denatured)topo.DNAintheSDS-K+precipitatecanberecoveredandshowntobeallnicked(topoIreactions)oramixtureofnicksanddoublestrandbreaks(topoII),asexpectedfromwhatweknowofthereactionmechanism. TheSDS-K+methodisparticularlyusefulforquantifyingtopoDNAcovalentcomplexesto:1)identifytopoIorIIinhibitorsorpoisonsand2)tofacilitatecomparisonofdifferentinhibitorsintheirabilitytoincreasethelifetimeofthecleavagecomplex.

                        CountPrecipitate                                                       CountPrecipitate