Formation and resolution of DNA catenanes by DNA gyrase.

We have discovered that DNA gyrase interlocks duplex DNA circles to form catenanes and resolves catenanes into component monomers. The reactions were inhibited by novobiocin and oxolinic acid and required ATP, Mg++ and spermidine. DNA sequence homology is not involved in catenation, since hybrid catenanes were formed efficiently between supercoiled phi X174 and Col E1 DNA. Strikingly different results were obtained with native and relaxed Col E1 DNA substrates. Up to 50-60% of input native DNA was converted into oligomeric catenanes, predominantly dimers and trimers. Relaxed substrates were instead converted into vast interlocked networks and were occasionally knotted. Optimal catenation occurred only in the narrow range of 20-35 mM KCl; increased ionic strength blocked catenation completely but activated the back reaction of decatenation. Gyrase resolved both the oligomeric catenanes and interlocked networks it produced, as well as naturally occurring catenanes. These results imply that the mechanism of gyrase involves a transient double-strand break and passage of a DNA segment through the resulting gap. Gyrase is representative of a general class of enzymes, found in both procaryotic and eucaryotic cells, that facilitate diffusion of duplex DNA segments through each other and may thereby solve topological problems arising from the replication, recombination and condensation of DNA.

Duke Scholars

Author Kenneth N. Kreuzer Biochemistry