The helical structure of double-stranded DNA poses a number of topological problems when the DNA duplex is transcribed and replicated. DNA topoisomerases resolve these problems by catalyzing changes in DNA topology through a mechanism involving the transient breakage and rejoining of the DNA. Two types of DNA topoisomerases have been identified on the basis of the number of DNA strands they transiently cleave; type l enzymes break and rejoin one strand while ll enzymes cleave both strands. In eukaryotes, each enzyme is highly conserved in terms of amino acid sequence and mechanism of action. The significance of the in vivo function of these enzymes is implicit not only in their conservation in all organism studied to date, but also as they constitute the cellular targets of a number of therapeutically important antitumor drugs. Increasing evidence supports a role for eukaryotic DNA topoisomerase l in transcription, replication and recombination. The enzyme presumably acts to relive the torsional tension in the DNA which results from the translocation of a transcription or replication complex along the DNA double helix. Supercoiling as a result of transcription has been demonstrated in vivo, however, there has yet to be a detailed analysis of the generation of localized domains of supercoiling in response to gene transcription. Moreover, a clear understanding of the effects of local changes on transcription initiation and elongation and the role that DNA topoisomerase l plays in such processes has not been forthcoming. This application proposes to address the function of yeast DNA topoisomerase l by first determining the domains and specific amino acids sequences of the enzyme which are essential for activity in vivo. In addition, a site-specific DNA topoisomerase will be constructed which will preferentially relax supercoiled DNA in the vicinity o f a specific DNA sequence. This will be accomplished by fusing DNA topoisomerase l mutants with the yeast GAL4 protein and with E. coli tus protein, both of which bind with high affinity to a well defined DNA sequence. This sequence- directed enzyme will be used in a series of experiments in yeast to directly study the generation of torsional strain in specific regions of the DNA in response to transcription in vivo. Furthermore, this construct will be used to define the relationship between DNA topology and transcription initiation and elongation.
