Cranking Up DNA, One Gyration at a Time

The mechanism of DNA gyrase, which supercoils bacterial DNA.

Imagine that you have a garden hose that is thirty miles long. How would you keep it from getting tangled? That is unlikely to be easy. Now add randomly placed heavy machinery that actively twists that hose as it travels / pulls along, causing it to wind up ahead, and unwind behind. And that machinery can be placed in either direction, often getting into head-on conflicts, not to mention going at quite different speeds. That is the problem our cells have, managing their DNA. 

They use a set of topoisomerases to manage the topology of DNA- that is, its twist-i-ness. One easy method is to nick the DNA on one of its two strands, allowing it to relax by spinning around the remaining phosphate bond, before resealing it back to a double strand and sending it on its way. But what if you encounter coils or knots that can't be resolved that way? The next level is to cut one entire DNA molecule, not just one side/strand of it, and pass the conflicting one though it. All organisms contain topoisomerases of both kinds, and they are essential.

How DNA gets twisted. While most topoisomerases relax DNA (top) to resolve the many twisty problems posed by transcription and replication, gyrase increases twist by grabbing and holding a quasi-positive twist, then cutting and resolving it, as shown at bottom.

Bacteria have an additional enzyme that we do not have, called gyrase, to crank up the supercoiling of their DNA, to make it easier to open for transcription. Gyrase works just like a type II topoisomerase that cuts a double-stranded DNA and lets another DNA through, but it does so in a special way that puts a twist on the DNA first, so instead of relaxing the DNA, it increases the stress. How exactly that works has been a bit mysterious, though gyrases and the general principles they operate under have been clear for decades. Gyrase uses ATP, and grabs onto two parts of a DNA molecule, one of which is pre-twisted into coil, after which one is cut and the other passed through to create a change (-2) in the twisting number of that DNA.

A general model of gyrase action. The G segment of DNA is firmly held by the gyrase dimer in the center.  The same DNA is forcibly twisted about, around the pinwheel structures, and bent back around to enter through the N-gate (as the T segment). Then, the N gate closes, paving the way for the G-segment to be cut and separated (step 3). ATP is the energy source behind all this structural drama. The T-segment then passes through the cut, enters the C-gate, and the cycle is complete.

A recent paper determined the structure of active gyrase complexes, and was able to trace the pre-twisted conformation. This, combined with a lot of past work on the ATPase and cleavage functions of gyrase, allows a reasonably full picture of how this enzyme works. It is a symetric dimer of a two-subunit protein, so there are four protein chains in all. There are three major regions of the full structure. The N-gate at top where one segment (the T-segment) of DNA binds, then the central DNA gate, where the other (G-segment) DNA binds and is later cut to let the T-segment through, and the C-gate, where the T segment ends up and is released at the end of the cycle. 

Focus on the pinwheel structure that dramatically pre-twists the DNA around between the G and T segments, pre-positioning the complex for strand passage and increased supercoiling.

The magic is that the T-segment and the G-segment of DNA are parts of the same DNA molecule, by being wrapped around the ears of the protein, which are also called pinwheels. That is what the newest structure solves in greatest detail. These pinwheels essentially allow the enzyme to yank an otherwise normal DNA strand into a pre-knotted (positive supercoil) form that, when cut and resolved as shown, results in a negative increment of supercoiling or twist. If they mutated the pinwheels away, the enzyme could still hold, cut, and relax DNA, but it could not increase its supercoiling. It is the ability of the pinwheel structures to set up a pre-twisted structure onto the DNA that makes this enzyme a machine to increase negative supercoiling, and thus ease other DNA transactions. 

Topoisomerase enzymes through evolution, from gyrase (left) to human topoII on the right. Note how the details of the protein structure are virtually unrecognizable, while the overall shape and DNA-binding stays the same.

Bacteria also have more normal type II topoisomerases that cut DNA merely to relax it, so one might wonder how these two enzymes get along. Well, gyrase is responsible for the overall negative supercoiling of the bacterial genome, while the other topoisomerases have more localized roles to relieve transient knots and over-twisting. Indeed, if you negatively twist DNA enough, you can separate its strands entirely, which is not usually desirable. Further research shows that too much of either topoisomerase is lethal, and that they are kept in balance by transcriptional controls over the amount of each topoisomerase. This suggests a futile cycle of DNA winding and unwinding, as the optimal condition in bacterial cells when both are present in just the right amounts. 

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