Mutational analysis of a type II topoisomerase cleavage site: distinct requirements for enzyme and inhibitors.
We have analyzed the DNA sequence requirements for cleavage of a 30 bp oligonucleotide that contains a strong bacteriophage T4 type II topoisomerase site. A novel method was used to generate substrates with each of the four nucleotides at 10 positions surrounding the cleavage site, and mutant substrates were also prepared for the four internal positions of the staggered cleavage site. The substrates were tested for cleavage in the presence of several inhibitors that induce enzyme-mediated cleavage: four antitumor agents of different classes (an aminoacridine, a substituted anthraquinone, an ellipticine derivative and an epipodophyllotoxin) and one antibacterial quinolone. At eight nucleotide positions flanking the cleavage site, the same preferred bases were found regardless of which inhibitor was present. These preferred bases show dyad symmetry with respect to the cleavage site, indicating that both protomers of the topoisomerase homodimer interact with DNA in an analogous manner. In addition, we found that the preferred bases on the 5' side of each cleaved phosphodiester bond are highly specific to the inhibitor used in the cleavage reaction. These results strongly suggest that the inhibitors interact directly with the DNA bases at the cleavage site, placing the inhibitor binding site precisely at the site of DNA cleavage.
Department of Microbiology and Immunology, Duke University Medical Center, Durham, NC 27710.
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D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000378:metabolism; D013379:Substrate Specificity; D059005:Topoisomerase II Inhibitors
D004279:DNA, Viral; D009841:Oligonucleotides; D059005:Topoisomerase II Inhibitors; D004250:DNA Topoisomerases, Type II