Cleavage of DNA by mammalian DNA topoisomerase II.
Using the P4 unknotting assay, DNA topoisomerase II has been purified from several mammalian cells. Similar to prokaryotic DNA gyrase, mammalian DNA topoisomerase II can cleave double-stranded DNA and be trapped as a covalent protein-DNA complex. This cleavage reaction requires protein denaturant treatment of the topoisomerase II-DNA complex and is reversible with respect to salt and temperature. The product after reversal of the cleavage reaction remains supertwisted, suggesting that the two ends of the putatively broken DNA are held tightly by the topoisomerase. Alternatively, the enzyme-DNA interaction is noncovalent, and the covalent linking of topoisomerase to DNA is induced by the protein denaturant. Detailed characterization of the cleavage products has revealed that topoisomerase II cuts DNA with a four-base stagger and is covalently linked to the protruding 5'-phosphoryl ends of each broken DNA strand. Calf thymus DNA topoisomerase II cuts SV40 DNA at multiple and specific sites. However, no sequence homology has been found among the cleavage sites as determined by direct nucleotide-sequencing studies.
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D010957:Plasmids; D013539:Simian virus 40 / Q000235:genetics
D004279:DNA, Viral; D004264:DNA Topoisomerases, Type I