RelaxedpHOT-1DNAProductDescription
RelaxedpHOT-1issuitedtoassayingthesupercoilingactivityofDNAgyrase. ItisalsousefulforassayingalterationsinDNAlinkingnumberforassessingintercalation.  RelaxedpHOT-1DNAispreparedusinghighpuritytopoIrelaxationreactions,followedbyinactivationandre-purificationoftherelaxedproduct. TopoGENstaffthenverifytherelaxationstatusofeachlotandvalidatesthatthesubstratecanbesupercoiledbyE.coliandS.aureusDNAgyrase.

TheplasmidpHOT1containsthehighaffinitytopoIcleavagesite(thehexadecamericsequencedescribedbyWestergaardandco-workers,seeBonvenetal.,1985)thatisderivedfromthetetrahymenaribosomalgenerepeat. ThisconstructisusefulasatopoIcleavagesubstratesincetheenzymeefficientlycleavesatthehexadecamericsiteintheabsenceofcamptothecin. ThisallowstheinvestigatortorigorouslyevaluatenewpotentialtopoIinhibitorsbecauseasinglebackgroundcleavageisabuilt-incontrol;iftheagentstimulatescleavageatthisand/oranyothersequenceinthefragmentthatcontainsthehexadecamericsite,prominentcuttingisreADIlyrevealed. Typically,itisdifficulttotraptopoIcleavagesonDNA;onemustusemoreenzymesincecleavagecomplexesconsumetheenzymestoichiometrically. Forthisreason,higherlevelsofenzymearerequiredforanalysisofDNAcleavages. Thefragmentcanbeisolatedfromthepolylinkerusingthesitesshownbelow(protocolsinManiatiscloningbook),endlabeledandusedasacleavagesubstrate.


Reference:
Bonvenetal.,Cell41:541(1985)

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