TopoGENprovidespurifiedHumanTyrosylDNAPhosphodiesterase(Tdp1)purifiedtohomogeneitytoasingle68kDabandonSDS-PAGE. Tdp1iscatalyticallyactiveandwillremoveaterminal3’phosphotyrosylgroupfromaspecificallydesignedoligotargetsite(oligo-Tyr). Additionally,TopoGENscientistshavedemonstratedthatTdp1willreleasetopoIcovalentlyattachedtogenomicDNAisolatedfromcellstreatedwithcamptothecinusingTopoGEN’sInVivoLinkKit(TG1021). Tdp1isthoughttobeimportantinrepairoftopo/DNAadductsthatformfollowingtopo-activedrugtherapy;therefore,thereisconsiderableinterestinidentifyingdrugsthattargetTdp1. Suchdrugsmayworkwellincombinationwithtopotargetingstrategies.

Figure1. SDS-PAGEofTopoGENPurifiedTdp1. Thegelwasintentionallyoverloadedwith20ugofprotentorevealminorbreakdownproducts.

UnitDefintion:
OneunitofTdp1canreleasetheterminalTyrosinefrom20ngofsubstrate(oligo-Tyr)in30minat37°C.

Tdp1Reaction:
ReleaseoftopoIfromthecovalentcomplexisshownbelow.

Tdp1Assays:
BiochemicalassaysforTdp1canbecarriedoutusinganoligonucleotidewitha3’terminaltyrosineresidue. Releaseoftyrosinefroma5’32PendlabeledoligoiseasilymeasuredusingadenaturingpolyacrylamidegelorDNAsequencinggels. TopoGENProvidesakitforTdp1assay(TG1004)thathaskeyreagentsandcontrols/protocolsforsettingupacompleteassayforTdp1.

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