S.aureusDNAGyraseProductBackground
HighlypurifiedS.aureusDNAgyraseisatypeIItopoisomeraseencodedbytwogenesGyrAandGyrBfromstaphylococcusaureus. DNAgyraseisanessentialtopoisomerasethatsupercoilsDNAthroughaprocessofstrandbreakage/resealingandDNAwrapping(3). AsatypeIIenzyme,gyraseisuniqueinitsABIlitytonegativelysupercoilarelaxedplasmidDNAsubstrate. S.aureusDNAgyraseisalsothetargetforquinolone-basedantibacterialagentswhichactbysubvertingtheenzymeintoaDNAdamagingagent. TopoGENofferspurifiedDNAgyrasefromthegrampositivebacterium,Staphylococcusaureusforuseinallaspectsofdrugdevelopmentandscreeningassays. TheenzymeisveryactiveindecatenatingandsupercoilingKDNA(TG2013)butwillalsonegativelysupercoilrelaxed plasmidDNA(TG2037). TheS.aureusenzymeisaheterotetramer ofGyrA2GyrB2andispurifiedasHistaggedsubunitsthatarereassembledtomakeactiveenzyme(4).

S.aureusDNAGyraseQualityControlTests
DNAgyrasesubunitswereclonedoverexpressedandpurifiedusingtheproprietarycompanymethods. AsinglebandonSDS-PAGEwasdetectedbyCBstainingforeachsubunit. CrosscontaminationbytopoIwasassessedbyassayingforrelaxationofsupercoiledDNAunderconditionsoptimizedfortypeIactivity. Undertheseconditions,after2hoursofincubationwithsupercoiledplasmidDNA,norelaxationproductsweredetectable.

AtestfornucleasecontaminationwascarriedoutbyassayingfortheformationoflinearKDNAandlinearplasmidDNA.  Incubationsof1µgofcatenatedKDNAorsupercoiledDNA(4hrs.at37°Cinthepresenceof10mMMgCl2)wereperformed. LinearDNAorbreakdownproductswerenotgeneratedundertheseconditions.

Thesubunitsarebetterthan95%purebaseduponSDS-PAGE.Neithersubunitisactiveintheabsenceoftheotherandneithersubunitdisplaysnucleolyticactivity. Thesedatashowthathost(E.coli)isnotcontributingtotheactivityofindividualsubunitsof S.aureusgyrAorB. ThiswasconfirmedbyWesternblottingprobingswithanti-GyrAIgGspecifictoE.coli(datanotshown).

AssayConditions
TopoGENprovidessamplesof*threebuffersthatareusedtoassayS.aureusDNAGyrase. Thebuffersare:
5XAssayStock(0.5mlprovided)containsthefollowingsolutes:

• 375mMTris-HCl(pH7.5)
• 37.5mMMgCl2
• 37.5mMDTT
• 0.375mg/mlBSA
• 150mMKCl
• 10XATPStock: 20mMATP(0.25mL). Enzymerequires2.0mMATPFINALCONCENTRATION
• 5XPotassiumGlutamateStock: 2.5M(0.25mL). Enzymerequries500mMK-GluFINALCONCENTRATION.

*Note:ThesebuffersareprovidedtoallowthecustomertoestablishaworkingcontrolfortheGyraseassay. Additionalbuffermayberequired. Storeallbuffersat-20°C.

Assaysshouldbeperformedinafinalvolumeof20ulbyaddingappropriateamountsofbuffers,DNAenzyme(addenzymelasttoinitiatetheassay). Typicalassaywouldbe:

(Enzymedilutedtoworkingconcentrationusingdilutionbufferortitrateas2foldserialdilutions.)
Incubate30min37°C,stopbyadditionofSDSto1%(notincluded);addBromophenolblue/glycerolloADIngdye,run1%agarosegelwithrelaxed/supercoiledMarkers. Optional: digestwithproteinaseKandrepurifyDNAbyphenolextraction. GelsshouldbestainedwithEthidiumBromide(0.5ug/ml20-30min),destainedinwaterfor15-30minatroomtemperatureandphotodocumented.

DilutionBuffer
Ifnecessary,dilutetheenzymeusingthefollowingbuffer(1x)suppliedwiththeenzyme.
50mMTris-HCl[pH7.5],100mMNaCl,2mMb-DTT,1mMEDTA,10%glycerol.

ModificationsoftheGyraseAssay: DetectingDNAcleavages.
GyraseisatargetforseveralantibioticsthatinducetheenzymetocleavetheplasmidDNAsubstrate(3,5).  Quinolonedrugsareknowntoinducecleavagesconcurrentwithcovalentcomplexformation;thus,todetectsuchcomplexesindrugscreeningexperiments,itisessentialthatproteinaseKdigestionsbecarriedoutandthatreactionproductsrepurifiedbyphenol/chloroformextraction. Additionally,theconditionsthatareoptimalforcleavagedetection,differslightlyfromthoseoptimizedforcatalyticassays. ForDNAGyrase,wefindthatreducedpotassiumglutamate(200mMfinalconcentration)isidealforcleavagecomplexformation. 

ToresolvenickedandlinearDNAcleavages,werecommendrunning1%agarosegelscontaining0.5u/mlEBinthegelandrunningbuffer. Destainfor15minpriortophotodocumentingtheresults. ItisessentialtoincludelinearDNAmarkersinthesegels.

• Distilledwater-8ul
• 5XAssaystock-4ul
• 10XATPStock-2ul
• 5XK-GluStock-4ul
• 0.1ugsupercoiledPlasmidDNA-1ul
• Purifiedenzyme(variable)-1ulAddedlast
  • The1xcleavagebuffercontains:
  • 75mMTris-HCl(pH7.5)
  • 7.5mMMgCl2
  • 7.5mMDTT
  • 2mMATP
  • 75ug/mlBSA
  • 30mMKCl
  • 200mMK-Glu

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.

PengH,MariansK:DNATopoisomeraseProtocolsVolumeI1999.p.163TGVi

WangJ:DNATopoisomerases. Ann.Rev.Biochem1996,65:635-692

美国TopoGEN公司成立于1991年，是一家诊断公司，由一批在拓扑异构酶领域工作和在基础、临床、应用及相关方面研究经验丰富的研究者创建，为以药物开发为基础的拓扑异构酶和机制研发人员提供创新产品和服务。药物筛选试剂盒可以分析拓扑异构酶活性药物，用于癌症化疗和抗菌方面。TopoGEN公司还能提供原核和真核化验拓扑异构酶 (I型，II，IV和旋转酶)检测试剂盒，体外体内诊断均可应用。TopoGEN公司还提供DNA载体、DNA裂解目标、拓扑异构酶抑制剂、拓扑异构酶抗体和相关试剂盒等完整产品线。