Mechanistic studies of eukaryotic type II DNA topoisomerase and its interaction with topo II-targeting drugs

Type II DNA topoisomerase is an essential enzyme conserved throughout evolution. It is dimeric in structure and catalyzes the ATP-dependent passage of a DNA segment through a transient double-strand break in another segment, thus changing the higher order structure of DNA molecules. Beyond its physiological functions in nearly all aspects of DNA metabolism, topoisomerase II is the target for some of the most active and widely prescribed anti-microbial and anti-tumor drugs.;The focus of my study has been the mechanistic studies of eukaryotic topoisomerase II, specifically the conformational changes of the protein during its catalysis and its interactions with various topoisomerase II-targeting drugs. Site-specific mutagenesis was used to identify an essential residue, Lys 359, in the ATP-dependent reactions of Drosophila topoisomerase II. The biochemical characterization of the mutations at this residue has indicated that substituting Lys359 with either Gln or Glu greatly diminished the strand passage activity and the DNA-dependent ATPase activity of topoisomerase II. Furthermore, the mutations have altered the ATP-induced conformational changes of the enzyme. Our data suggest that Lys359 is critical for the catalytic activity of topoisomerase II and may have an important function in the ATP signaling process.;To characterize the biochemical properties of individual domains of eukaryotic topoisomerase II, truncation studies were carried out on two sets of eukaryotic topoisomerase II constructs that contain different domains of the protein. The results have shown that both core domain and the ATPase domain truncation mutants retain their intrinsic biochemical activities respectively. I have also studied the interactions between each truncated topoisomerase II and a novel class of anti-tumor drugs, bisdioxopiperazine (ICRF compounds). ICRF drugs can induce the formation of a clamp complex between circular DNA and the core domain truncation mutants, suggesting the drugs promote the closure at B' interface of the core domain. In addition, ICRF drugs can inhibit the ATPase activities of the ATPase domain mutants, indicating a direct interaction between this domain and the drug. These results have indicated that the drug can interact with both sets of truncation mutants.