DNA gyrase-mediated wrapping of the DNA strand is required for the replication fork arrest by the DNA gyrase-quinolone-DNA ternary complex

The ability of DNA gyrase (Gyr) to wrap the DNA strand around itself allows Gyr to introduce negative supercoils into DNA molecules, It has been demon strated that the deletion of the C-terminal DNA-binding domain of the GyrA subunit abolishes the ability of Gyr to wrap the DNA strand and catalyze th e supercoiling reaction (Kampranis, S. C., and Maxwell, A (1996) Proc. Natl , Acad Sci, U.S. A. 93, 14416-14421), By using this mutant Gyr, Gyr (A59), we have studied effects of Gyr-mediated wrapping of the DNA strand on its r eplicative function and its interaction with the quinolone antibacterial dr ugs. We find that Gyr (A59) can support oriC DNA replication in vitro, Howe ver, Gyr (A59)-catalyzed decatenation activity is not efficient enough to c omplete the decatenation of replicating daughter DNA molecules, As is the c ase with topoisomerase TV, the active cleavage and reunion activity of Gyr is required for the formation of the ternary complex that can arrest replic ation fork progression in vitro. Although the quinolone drugs stimulate the covalent Gyr (A59)-DNA complex formation, the Gyr (A59)-quinolone-DNA tern ary complexes do not arrest the progression of replication forks. Thus, the quinolone-induced covalent topoisomerase DNA complex formation is necessar y but not sufficient to cause the inhibition of DNA replication, We also as sess the stability of ternary complexes formed with Gyr (A59), the wild typ e Gyr, or topoisomerase IV. The ternary complexes formed with Gyr (A59) are more sensitive to salt than those formed with either the wild type Gyr or topoisomerase IV. Furthermore, a competition experiment demonstrates that t he ternary complexes formed with Gyr (A59) readily disassociate from the DN A, whereas the ternary complexes formed with either the wild type Gyr or to poisomerase IV remain stably bound. Thus, Gyr-mediated wrapping of the DNA strand is required for the formation of the stable Gyr-quinolone-DNA ternar y complex that can arrest replication fork progression.