Site-specific Cleavage of a DNA Hairpin by Topoisomerase II. DNA Secondary Structure As a Determinant of Enzyme Recognition/cleavage

To further define the nucleic acid determinants that govern the recognition of DNA by topoisomerase II, the ability of the enzyme to cleave a 51-base oligonucleotide that contained a centrally located 19-base hairpin was characterized. Topoisomerase II cleaved the 51-mer in a site-specific fashion, within the hairpin, one nucleotide from the 3'-base of the stem. Protein denaturants were not required to trap cleavage products. Although the sequence of the oligonucleotide influenced levels of enzyme-mediated DNA scission, it did not affect the spatial location of cleavage. DNA scission required a double-stranded/single-stranded junction at the 3'-base of the hairpin and a tail (either single- or double-stranded) at least 8 bases in length on the 5'-side. Cleavage was not observed when base-pairing within the oligonucleotide was eliminated or when the hairpin was extended to produce a completely double-stranded substrate. Finally, the enzyme displayed a size constraint for both the stem and loop structures of the hairpin. These results indicate that topoisomerase II is capable of recognizing secondary structure within nucleic acids and identifies the first secondary structure-specific DNA recognition/cleavage site for the type II enzyme.