Abstract
AbstractTopoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of topoisomerases: the type I enzymes, which make single‐stranded cuts in DNA, and the type II enzymes, which cut and pass double‐stranded DNA. In this unit, an in vitro assay for topoisomerase I activity based on relaxation of supercoiled DNA by that enzyme is described. This is followed by an assay for topoisomerase II based on the decatenation of double‐stranded DNA. In conjunction with these assays, the preparation of mammalian cell extracts for assaying topoisomerase activity is described. Procedures are also detailed for the assay of topoisomerase covalent complexes in vivo and for measuring DNA cleavage caused by topoisomerase I and topoisomerase II in vitro. A related electrophoretic method for mapping topoisomerase II cleavage sites is finally included.
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