DNA topoisomerases have been the subject of extensive research in recent years, particularly in view of the fact that many clinically relevant antibiotics and antitumor drugs specifically target these enzymes. Escherichia coli DNA topoisomerase III (Topo III) is a novel topoisomerase in that the preferred reaction of the enzyme, in vitro, appears to be the separation of intertwined chromosomes rather than the modulation of the superhelical density of chromosomal DNA. The experiments detailed in this proposal are designed to achieve two specific goals: to provide an understanding of the role of Topo III in DNA metabolism, and to provide information as to the functional organization of polypeptide. Using a novel mutagenesis and selection procedure, it is proposed to isolate and characterize, both in vitro and in vivo, mutations that eliminate or alter the activity of the topoisomerase. Specifically, it has been proposed to: 1) obtain a series of missense, nonsense, and temperature sensitive (ts) mutations within the Topo III polypeptide. 2) purify the mutant Topo III polypeptides and analyze the in vitro properties of enzymes. 3) organize the mutations into specific classes that are defective in different aspects of the reactions catalyzed by topoisomerases in an effort to define structural domains of the enzyme (i.e., DNA binding domain, strand passage domain). 4) replace the wild type copy of the gene encoding Topo III (topB) with mutant genes of the specific classes of mutations (including a temperature sensitive allele of topB). 5) attempt to correlate the in vitro biochemical properties of the mutant enzymes with specific structural changes within the polypeptide.
