In the presence of subunit A inhibitors DNA gyrase cleaves DNA fragments as short as 20 bp at specific sites.
A key step in the supercoiling reaction is the DNA gyrase-mediated cleavage and religation step of double-stranded DNA. Footprinting studies suggest that the DNA gyrase binding site is 100-150 bp long and that the DNA is wrapped around the enzyme with the cleavage site located near the center of the fragment. Subunit A inhibitors interrupt this cleavage and resealing cycle and result in cleavage occurring at preferred sites. We have been able to show that even a 30 bp DNA fragment containing a 20 bp preferred cleavage sequence from the pBR322 plasmid was a substrate for the DNA gyrase-mediated cleavage reaction in the presence of inhibitors. This DNA fragment was cleaved, although with reduced efficiency, at the same sites as a 122 bp DNA fragment. A 20 bp DNA fragment was cleaved with low efficiency at one of these sites and a 10 bp DNA fragment was no longer a substrate. We therefore propose that subunit A inhibitors interact with DNA at inhibitor-specific positions, thus determining cleavage sites by forming ternary complexes between DNA, inhibitors and DNA gyrase.
Gmünder
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F. Hoffmann-La Roche Ltd, Pharmaceutical Research Preclinical Infectious Diseases, CH-4070 Basel, Switzerland. hans.gmuender@roche.com
Kuratli
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K
Keck
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D001562:Benzimidazoles / Q000737:chemistry / Q000494:pharmacology; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002939:Ciprofloxacin / Q000737:chemistry / Q000494:pharmacology; D016576:Fleroxacin / Q000737:chemistry / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D015394:Molecular Structure; D010957:Plasmids / Q000378:metabolism*; D011743:Pyrimidines / Q000737:chemistry / Q000494:pharmacology; D059005:Topoisomerase II Inhibitors
D001562:Benzimidazoles; D011743:Pyrimidines; D059005:Topoisomerase II Inhibitors; C085885:Ro 47-3359; D002939:Ciprofloxacin; D008274:Magnesium; D016576:Fleroxacin; D002118:Calcium