Eukaryotic topoisomerase II. Characterization of enzyme turnover.
While the binding of adenyl-5'-yl imidodiphosphate (App(NH)p) to Drosophila melanogaster topoisomerase II induces a double-stranded DNA passage reaction, its nonhydrolyzable beta,gamma-imidodiphosphate bond prevents enzyme turnover (Osheroff, N., Shelton, E. R., and Brutlag, D. L. (1983) J. Biol. Chem. 258, 9536-9543). Therefore, this ATP analog was used to characterize the interactions between Drosophila topoisomerase II and DNA which occur after DNA strand passage but before enzyme turnover. In the presence of App(NH)p, a stable post-strand passage topoisomerase II-nucleic acid complex is formed when circular DNA substrates are employed. Although noncovalent in nature, these complexes are resistant to increases in ionic strength and show less than 5% dissociation under salt concentrations (greater than 500 mM) that disrupt 95% of the enzyme-DNA interactions formed in the absence of App(NH)p or under a variety of other conditions that do not support DNA strand passage. These results strongly suggest that the process of enzyme turnover not only regenerates the active conformation of topoisomerase II but also confers upon the enzyme the ability to disengage from its nucleic acid product. Experiments with linear DNA molecules indicate that after strand passage has taken place, topoisomerase II may be able to travel along its DNA substrate by a linear diffusion process that is independent of enzyme turnover. Further studies demonstrate that the regeneration of the enzyme's catalytic center does not require enzyme turnover, since topoisomerase II can cleave double-stranded DNA substrates after strand passage has taken place. Finally, while the 2'-OH and 3'-OH of ATP are important for its interaction with Drosophila topoisomerase II, neither are required for turnover.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000818:Animals; D003570:Cytidine Triphosphate / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004270:DNA, Circular / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004331:Drosophila melanogaster / Q000201:enzymology*; D004587:Electrophoresis, Agar Gel; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D015636:Magnesium Chloride; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology
D004270:DNA, Circular; D004278:DNA, Superhelical; D015636:Magnesium Chloride; D000266:Adenylyl Imidodiphosphate; D012967:Sodium Dodecyl Sulfate; D000244:Adenosine Diphosphate; D003570:Cytidine Triphosphate; D004250:DNA Topoisomerases, Type II; D008274:Magnesium