Topoisomerase assays.
Topoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of topoisomerases: type I enzymes, which make single-stranded cuts in DNA, and type II enzymes, which cut and pass double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. The protocols described in this unit are for assays used to assess new chemical entities for their ability to inhibit both forms of DNA topoisomerase. Included are an in vitro assay for topoisomerase I activity based on relaxation of supercoiled DNA, and an assay for topoisomerase II based on the decatenation of double-stranded DNA. The preparation of mammalian cell extracts for assaying topoisomerase activity is described, along with a protocol for an ICE assay to examine topoisomerase covalent complexes in vivo, and an assay for measuring DNA cleavage in vitro.
Molecular Pharmacology Department, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
John L
JL
Nitiss
Eroica
E
Soans
Anna
A
Rogojina
Aman
A
Seth
Margarita
M
Mishina
D000818:Animals; D000936:Antigen-Antibody Complex / Q000378:metabolism; D002457:Cell Extracts; D002462:Cell Membrane / Q000276:immunology; D002478:Cells, Cultured; D053837:DNA Cleavage / Q000187:drug effects; D026942:DNA Topoisomerases / Q000378:metabolism* / Q000494:pharmacology; D043864:DNA, Catenated / Q000187:drug effects* / Q000378:metabolism; D004278:DNA, Superhelical / Q000187:drug effects* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D057075:Enzyme Assays / Q000379:methods*; D006801:Humans; D051379:Mice; D010957:Plasmids / Q000494:pharmacology; D059003:Topoisomerase Inhibitors / Q000494:pharmacology
D000936:Antigen-Antibody Complex; D002457:Cell Extracts; D043864:DNA, Catenated; D004278:DNA, Superhelical; D059003:Topoisomerase Inhibitors; D026942:DNA Topoisomerases