An integrated biochemical and genetic approach will be used to investigate the mechanism, specificity, intracellular targeting and multiple functions of the human type I topoisomerase (topo I). With the availability of milligram quantities of pure topo I and high titer affinity purified antibodies against the protein, a number of biologically important issues can be investigated.
The specific aims are (i) to produce sufficient quantities of different forms of pure topo I with and without bound duplex oligonucleotides for crystallographic studies, (ii) to use a mutant form of topo I containing a substitution of phenylalanine for the active site tyrosine (Y723F mutant) to study how DNA sequence and DNA topology affect the binding of DNA by the protein, (iii) to use the ligation-mediated polymerase chain reaction to avoid the use of camptothecin in identifying topo I break sites associated with transcription and DNA replication in human cells, (iv) to determine the effects of overexpressing the wild type and Y723F mutant forms of topo I in human cells, and (v) to isolate cDNAs which encode proteins that specifically interact with topo I. The biochemical and crystallographic studies will elucidate the details of the catalytic mechanism of topo I as well as provide an understanding of the basis for the inhibitory effects of the anti-cancer drug, camptothecin. These studies will also clarify the relationship between the sequence requirements for the binding of the enzyme to DNA with the sequence requirements for topo I- mediated breakage of DNA after addition of denaturants. The overexpression studies in combination with the search for interacting partners of the protein should provide important insights into how the topo I associates with other proteins in the cell to provide the swivels required for DNA replication and transcription. Studies which address the involvement of topo I in illegitimate recombination may reveal a role for the enzyme in the genetic instability of cancer cells, especially colon carcinoma cells which exhibit elevated levels of topo I.
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