Nucleic Acids Research, 1994, Vol. 22, No. 9 1567-1575
© 1994
MOLECULAR BIOLOGY |
Evidence for a conformational change in the DNA gyrase–DNA complex from hydroxyl radical footprinting
Department of Biochemistry, University of Leicester Leicester LE1 7RH, UK
*To whom correspondence should be addressed
Received February 15, 1994. Revised March 25, 1994. Accepted March 25, 1994.
We have used the technique of hydroxyl radical footprinting to probe the complex between DNA gyrase and a 198 bp DNA fragment containing the preferred gyrase cleavage site from plasmid pBR322. We find that gyrase protects 128 bp from the hydroxyl radical with the central 13 bp (adjacent to the gyrase cleavage site) being most strongly protected. Flanking the central region are arms showing periodic protection from the reagent suggesting a helical repeat of 10.6 bp, consistent with the DNA being wrapped upon the enzyme surface. The presence of 5'-adenylyl-ß,
-imidodiphosphate or a quinolone drug causes alteration of the protection pattern consistent with a conformational change in the complex involving one arm of the wrapped DNA. The significance of these results for the mechanism of DNA supercoiling by gyrase is discussed.
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