Mutational analysis of a type II topoisomerase cleavage site: distinct requirements for enzyme and inhibitors.

Published

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Freudenreich, CH; Kreuzer, KN

Published Date

  • May 1993

Published In

Volume / Issue

  • 12 / 5

Start / End Page

  • 2085 - 2097

PubMed ID

  • 8387918

Pubmed Central ID

  • 8387918

International Standard Serial Number (ISSN)

  • 0261-4189

Language

  • eng

Conference Location

  • England