October 2, 2017

Unique Properties of DNA Gyrase for (+) Supercoil Processing

The twisting of DNA in the same or opposite direction of the turn of the double helix gives rise to (+) or (-) supercoiling, respectively. Supercoiling exerts a strain on the helix, leading to the formation of loops, knots, and tangles that can have a major impact on the ability of DNA processing enzymes to carry out their function. This is especially true with regard to (+) supercoils that form ahead of the replication or transcription machinery as a result of DNA unwinding. In bacteria, (+) supercoils are removed through the action of two enzymes, gyrase and topoisomerase IV. Both enzymes work by grasping a strand of double-stranded DNA, introducing a break in that strand, and then passing a second strand through the break before it is reconnected. Topoisomerase IV can remove (-) as well as (+) supercoils, whereas gyrase introduces (-) supercoils in relaxed DNA. This difference in (-) supercoil processing is associated with the ability of gyrase, but not topoisomerase IV to wrap the second strand of DNA before passing it through the break. Relatively little is known, however, about how the two enzymes process (+) supercoils, leading Vanderbilt Basic Sciences investigator Neil Osheroff and his graduate student Rachel Ashley to take a closer look. They discovered that gyrase removes (+) supercoils much faster than it introduces (-) supercoils, and that it does so in bursts of activity as fast as 107 supercoils/s. A mutant enzyme unable to wrap DNA exhibited markedly reduced efficiency in (+) supercoil processing, indicating that wrapping was required for both of gyrase’s primary functions. Topoisomerase IV also removed (+) supercoils more quickly than (-) ones, but it was much less efficient than gyrase at (+) supercoil processing. When gyrase or topoisomerase IV break the first bound strand of DNA, it becomes covalently bound to the enzyme, forming a cleavage complex. Failure to rejoin the ends of the DNA strand can lead to double strand breaks and ultimately the death of the cell. Quinolone antimicrobial agents work by stabilizing the cleavage complex. The researchers showed that in the presence of quinolones, gyrase forms fewer double strand breaks while processing (+) supercoiled DNA than (-) supercoiled DNA. For topoisomerase IV, there was little difference in break formation between the two substrates. The results suggest that gyrase is particularly well-suited to process (+) supercoils formed ahead of the replication or transcription machinery in terms of both the rapidity of the process and the low rate of double strand break formation. The work is published in the journal Nucleic Acids Research [R. E. Ashley, et al. Nuc. Acids Res., 45, 9611].