Type I topoisomerases are enzymes that alter the topology of DNA by transiently breaking and rejoining single DNA strands. The eukaryotic family of type I enzymes, including the ubiquitous nuclear topo I and the topo I of vaccinia virus, comprises a group of proteins with shared biochemical properties and catalytic mechanism. The involvement of these enzymes in such fundamental processes as DNA replication, genetic recombination, and transcription, as well as the fact that the cellular topo I is the target of a class of antitumor drugs of emerging clinical import (camptothecin and its congeners), mandates a more complete understanding of the mechanism of action of the type I topoisomerases, both in vivo and in vitro. The goal of this laboratory is to achieve such an understanding using vaccinia virus as a model system for a combined biochemical and genetic study of eukaryotic topo I function. Vaccinia is unique among eukaryotic DNA viruses in that it encapsidates within the infectious virus particle a type I topo that is similar in its enzymatic properties to the cellular counterpart. The vaccinia enzyme is distinguished from its cellular counterparts by its smaller size, Mr 32,000, and its resistance to camptothecin. The viral gene encoding the topo has been identified, and the enzyme has been overexpressed in active form in bacteria. Most important, the viral topoisomerase gene is essential for the replication of vaccinia in cell culture. Experiments proposed herein are designed to illuminate aspects of topo structure and function shared by members of the eukaryotic enzyme family while focusing in detail on the functional distinctions between viral and cellular enzymes. Three complementary lines of investigation are described. The first approach, a molecular genetic analysis, seeks to create a comprehensive structure-function map of the vaccinia enzyme, by combining various mutagenesis techniques with the ability to select genetically those mutant alleles that affect topo activity. This will include selection of conditionally defective temperature-sensitive alleles. In addition, chimeric topoisomerase molecules -part viral and part cellular - will be created (by gene fusion techniques) in order to map precisely the domains in the cellular and viral topoisomerase proteins that confer sensitivity vs resistance to camptothecin. The molecular determinants of sensitivity to this antineoplastic agent may be of considerable practical import, not only in future drug design, but in anticipation of the emergence of clinical resistance among patients to whom the drug may ultimately be given. The second avenue, largely biochemical, entails determination of the structure of the vaccinia topo via crystallography and examination, by cocrystallization with defined substrates, of how the enzyme interacts with its specific binding motif in DNA. The functional relevance of this sequence element to the ability of topoisomerase to catalyze rearrangements of nucleic acid sequences in vitro will also be studied. The third approach, genetically based, will be to isolate virus mutants affected conditionally in DNA topoisomerase activity so as to understand more fully the physiologic role of this enzyme in vivo.
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