title: Novobiocin affinity purification of a mitochondrial type II topoisomerase from the trypanosomatid Crithidia fasciculata.
abstract: A mitochondrial type II DNA topoisomerase (topoIImt) has been purified to near homogeneity from the trypanosomatid Crithidia fasciculata. A rapid purification procedure has been developed based on the affinity of the enzyme for novobiocin, a competitive inhibitor of the ATP-binding moiety of type II topoisomerases. The purified enzyme is capable of ATP-dependent catenation and decatenation of kinetoplast DNA networks as well as catalyzing the relaxation of supercoiled DNA. topoIImt exists as a dimer of a 132-kDa polypeptide. Immunoblots of whole cell lysates show a single predominant band that comigrates with the 132-kDa polypeptide, indicating that the 264-kDa homodimer represents the intact form of the enzyme. Localization of the enzyme within the single mitochondrion of C. fasciculata (Melendy, T., Sheline, C., and Ray, D. S. (1988) Cell, in press) suggests an important role for topoIImt in kinetoplast DNA replication.
authors.affiliation: Molecular Biology Institute, University of California, Los Angeles 90024.
authors.forename: T
authors.initials: T
authors.lastname: Melendy
authors.forename: D S
authors.initials: DS
authors.lastname: Ray
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002846:Chromatography, Affinity; D003421:Crithidia; D004250:DNA Topoisomerases, Type II / Q000302:isolation & purification*; D004278:DNA, Superhelical / Q000378:metabolism; D008970:Molecular Weight; D009675:Novobiocin / Q000378:metabolism*; D009690:Nucleic Acid Conformation
chemical_list: D004278:DNA, Superhelical; D009675:Novobiocin; D000255:Adenosine Triphosphate; D004250:DNA Topoisomerases, Type II