Abstract
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.
Highlights
DNA topoisomerases are enzymes that catalyze changes in the topology of DNA via a mechanism involving the transient breakage and rejoining of phosphodiester bonds in the DNA backbone
Our studies have shown that in the presence of ICRF-159, both mutants could trap circular DNA in a topological link, suggesting that the drug targets the core domain of the enzyme and promotes the intradimeric closure at the N-terminal interface in this protein
The ND406, core domain, and wild-type topo II were overexpressed in yeast cells and purified to over 90% in homogeneity based on the analysis of an SDS-polyacrylamide gel electrophoresis gel stained with Coomassie Blue dye (Fig. 2)
Summary
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 includes the regions homologous to the C-terminal one-third of the gyrB subunit (BЈ) and N-terminal two-thirds of the gyrA subunit (AЈ) This part of the gyrA subunit contains the active site tyrosine and is involved in breaking and rejoining the DNA backbone (reviewed in Ref. 5). Our studies have shown that in the presence of ICRF-159, both mutants could trap circular DNA in a topological link, suggesting that the drug targets the core domain of the enzyme and promotes the intradimeric closure at the N-terminal interface in this protein
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