ACRB-MUTATION LOCATED AT CARBOXYL-TERMINAL REGION OF GYRASE-B SUBUNITREDUCES DNA-BINDING OF DNA-GYRASE

Mutations that exhibit susceptibility to acriflavine have been isolate d and classified as acr mutations in Escherichia coil. We cloned the a crB gene, which has been identified as a mutation of the gyrB gene, an d found a double point mutation altering two consecutive amino acids ( S759R/R760C) in the COOH-terminal region of the gyrase B subunit, The mutant B subunit was found to associate with the A subunit to make the quaternary structure, and the reconstituted gyrase showed an 80-fold reduction of specific activity in DNA supercoiling assay; the sensitiv ity to acriflavine was not different in the same unit of wild-type and mutant gyrases. The mutant enzyme retained intrinsic ATPase activity, but DNA dependent stimulation was observed infrequently. A gel shift assay showed that acriflavine inhibited the DNA binding of gyrase, The acrB mutation also reduced significantly the DNA binding of gyrase bu t did not change the sensitivity to acriflavine, These results reveale d that the acrB mutation is related to the inhibitory mechanism of acr iflavine; and the acriflavine sensitivity of the mutant, at least in v itro, is caused mainly by reduction of the enzyme activity, Further, o ur findings suggest that the COOH-terminal region of the B subunit is essential for the initial binding of gyrase to the substrate DNA.