Calcium-promoted DNA cleavage by eukaryotic topoisomerase II: trapping the covalent enzyme-DNA complex in an active form.
The effects of calcium ions on interactions between Drosophila melanogaster topoisomerase II and DNA were assessed. Although the divalent cation could not support DNA strand passage, it was able to promote high levels of enzyme-mediated DNA cleavage. Moreover, sites of cleavage on plasmid pBR322 generated in calcium-promoted reactions were similar to those obtained in the presence of magnesium. When calcium-containing enzyme-DNA mixtures were treated with ethylenediaminetetraacetic acid, cleaved nucleic acids could be generated in the absence of sodium dodecyl sulfate (SDS) or other denaturing detergents. The product of this SDS-independent calcium-promoted reaction was a covalent topoisomerase II-DNA complex. Enzyme molecules trapped in such complexes were found to be kinetically competent. Therefore, calcium should be a valuable tool for studying the enzymology of topoisomerase II mediated DNA cleavage.
Osheroff
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Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Zechiedrich
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D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002413:Cations, Divalent; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism; D004331:Drosophila melanogaster / Q000201:enzymology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010957:Plasmids; D011485:Protein Binding
D002413:Cations, Divalent; D004269:DNA, Bacterial; D004250:DNA Topoisomerases, Type II; D008274:Magnesium; D002118:Calcium