SupercoiledpHOT-1DNAProductDescription
Thisplasmidisaperfectsubstrateforassayinganytopoisomerase. pHOT-1isrelativelysmall(<3KB) which is ideal for displaying intermediate topoisomers.  Since all topoisomerases display degenerate DNA binding and cleavage sequences, pHOT-1 will effectively support activity for a wide array of topoisomerase.  Supercoiled pHOT-1 is a scaled-down derivative of pBR322 and optimized for higher yields.  It has one additional feature for researchers who wish to assess human topo I cleavage at a single sequence: we have included the human topoisomerase I preferential binding site (the hexadecameric sequence described by Westergaard and co-workers, see Bonven et al., 1985) that is derived from the tetrahymena ribosomal gene repeat.  This construct is useful as a topo I cleavage substrate since the enzyme efficiently cleaves at the hexadecameric site in the absence of camptothecin.  This allows the investigator to rigorously evaluate new potential topo I inhibitors because a single background cleavage is a built-in control; if the agent stimulates cleavage at this and/or any other sequence in the fragment that contains the hexadecameric site, prominent cutting is reADIlyrevealed. Typically,itisdifficulttotraptopoIcleavagesonDNA;onemustusemoreenzymesincecleavagecomplexesconsumetheenzymestoichiometrically. Forthisreason,higherlevelsofenzymearerequiredforanalysisofDNAcleavages. Thefragmentcanbeisolatedfromthepolylinkerusingthesitesshownbelow(protocolsinManiatiscloningbook),endlabeledandusedasacleavagesubstrate.

SupercoiledpHOT-1DNAisincludedwithTopoIAssayKit,TopoIDrugKit,TopoIIDrugKit,andTopoIVDrugKit. 

Reference:
Bonvenetal.,Cell41:541(1985)