Analysis of a core domain in Drosophila DNA topoisomerase II. Targeting of an antitumor agent ICRF-159.
To investigate the biochemical properties of individual domains of eukaryotic topoisomerase (topo) II, two truncation mutants of Drosophila topo II were generated, ND406 and core domain. Both mutants lack the ATPase domain, corresponding to the N-terminal 406 amino acid residues in Drosophila protein. The core domain also lacks 240 amino acid residues of the hydrophilic C-terminal region. The mutant proteins have lost DNA strand passage activity while retaining the ability to cleave the DNA and the sequence preference in protein/DNA interaction. The cleavage experiments carried out in the presence of several topo II poisons suggest that the core domain is the key target for these drugs. We have used glass-fiber filter binding assay and CsCl density gradient ultracentrifugation to monitor the formation of a salt-stable, protein-clamp complex. Both truncation mutant proteins can form a clamp complex in the presence of an antitumor agent, ICRF-159, suggesting that the drug targets the core domain of the enzyme and promotes the intradimeric closure at the N-terminal interface of the core domain. Furthermore, the salt stability of the closed protein clamp induced by ICRF-159 depends on the presence and closure of the N-terminal ATPase domain.
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
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D000251:Adenosine Triphosphatases / Q000235:genetics; D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000235:genetics; D004270:DNA, Circular / Q000378:metabolism; D004330:Drosophila / Q000201:enzymology*; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D011487:Protein Conformation / Q000187:drug effects; D011929:Razoxane / Q000378:metabolism*; D013379:Substrate Specificity
D000970:Antineoplastic Agents; D004270:DNA, Circular; D011929:Razoxane; D000251:Adenosine Triphosphatases; D004250:DNA Topoisomerases, Type II