During the process of DNA replication and transcription, DNA strands are subjected to various topological conformations. Due to the intertwined nature of the DNA strands, they are strained throughout the process of replication and can become over-wound or under-wound from their natural state. These over or under wound DNA are then referred to as “supercoiled”. This effect is particularly seen ahead of the replication fork, where DNA strands can become highly overwound, generating large amounts of tension that can eventually prevent replication from continuing. Over-wound supercoils are referred to as “positive supercoils” while under-wound DNA coils are considered “negative supercoils”.
In order to compensate for this supercoiling phenomenon of DNA, a family of enzymes works to prevent damaging or disruptive supercoils. This family of Type II topoisomerases is specifically responsible for several functions in the replication and transcription processes, such as catalyzing the transport of one DNA segment through another, and unlinking sister chromosomes during replication. Regulating the amount of DNA supercoiling allowed throughout the replication, transcription and recombination phases is the topoisomerase family’s’ most unique role1). The type II topoisomerases function by catalyzing the passage of one strand of double-stranded DNA through an opening in a second, cleaved strand of DNA2). The strands are referred to as the transfer or T segment, and the gate or G segment respectively. Since type II topoisomerases can wind, unwind, unknot and unwind DNA, their cooperative activity is essential for maintenance of DNA topology. Topoisomerase IV, a member of the type II family, is one of several prokaryotic enzymes responsible for this regulation.
In prokaryotes, two structurally similar, but functionally unique Type II Topoisomerases enzymes are present; gyrase and topoisomerase IV, both of which are targets of quinolone antibiotic drugs3). The two enzymes share a remarkable amount of amino acid sequence similarity, but have distinct roles in topoisomerization4). Gyrase regulates transcription by creating negative supercoils in DNA strands that compact the strands together. Topoisomerase IV is more important in DNA replication, where it removes downstream positive supercoils, and unlinks sister chromosomes immediately after their synthesis. Topoisomerase IV is specifically suited to this task because it binds preferentially to positively supercoiled DNA, and doesn’t easily alter negative supercoils. Although it doesn’t preferentially unwind negative supercoils, topoisomerase IV does bind equally with them, implying that its chiral discrimination toward unwinding positive supercoils is not due to enzymatic binding efficiency5). In fact, topoisomerase IV preferentially binds any supercoiled DNA over relaxed DNA and this is a function more analogous to eukaryotic Type II topoisomerase enzymes than to other prokaryotic enzymes6). The limited action topoisomerase IV has when bound to negative supercoils prevents it from reversing the action of gyrase, which purposely induces negative supercoils to aid in transcription7). Unlike gyrase, topoisomerase IV doesn’t wrap DNA around itself or distort the helical path when it binds to DNA8). Under some conditions, topoisomerase IV is also able to substitute for the DNA relaxation activity of other enzymes, particularly topoisomerase I 9). All topoisomerase IV actions require the hydrolysis of ATP. Only topoisomerase IV is able to support the final replication stages and to allow for the separation of replicated daughter DNA strands10).
Recent studies regarding topoisomerase IV’s discrimination between negative and positive supercoils have determined that the enzyme is able to distinguish between “left handed” and “right handed” crossing patterns between crossing DNA strands. Topoisomerase IV specifically acts on left-handed crossovers, which happen to be found approximately 25 times more frequently in positive supercoils than in negative supercoils11). This rationale explains why topoisomerase IV demonstrates 20-fold preference toward positive supercoils. It is also suspected that topoisomerase IV is able to bind the transfer and gate segments when they are in an acute crossing angle, or less than 90 degrees, and this conformation occurs most often in positive supercoils12). Topoisomerase IV is also known to bind in a unique conformation when bound to positive supercoils, in contrast to its binding with negative supercoils13). Topoisomerase IV action involves the enzyme binding to DNA, and forming an equilibrium for the cleavage reaction. When the topoisomerase is then denatured, the DNA is cleaved. The rate of DNA cleavage is dependent on the equilibrium constant of the reaction14). While it is understood that topoisomerase IV must have a mechanism for distinguishing between positive and negative supercoils, the precise mechanism for its relaxation action is still speculative, but is known to depend on the ParC and ParE subunits that make up the holoenzyme.
Topoisomerase IV is encoded by the parC and parE genes and functional topoisomerase IV is a holoenzyme comprised of two ParC and two ParE subunits, denoted ParC2E2. The ParC subunit is 752 amino acids long and is positioned in a manner that allows it to act favorably with certain DNA geometries, enabling it to serve as a control of substrate specificity15). The ParC C-terminal domain of active topoisomerase IV is thought to contribute heavily to the unique functions of the enzyme in contrast to gyrase16). When the activity of topoisomerase enzymes lacking the ParC C-terminal domain was tested, they unwound both negative and positive supercoils with similar efficiency. This finding implies that the C-terminal domain of this subunit plays a crucial role in topoisomerase IV substrate specificity. Removing the C-terminal domain of ParC also influenced the decatenation activity of topoisomerase IV, decreasing it 100-fold17). The heterotetramer of topoisomerase IV also includes two subunits of ParE, a 630 amino acid domain, along with the two subunits of ParC. The ParE subunit is important in regulating the enzyme function. Only when the enzyme is bound to positive supercoils can the ParE subunits interact to facilitate unwinding18). Both the ParC and ParE subunits are necessary for functional Topoisomerase activity 19).
Along with topoisomerase IV's ability to unwind supercoiled DNA strands, it has a unique ability to unknot tangled DNA. Knotted DNA strands have a hugely detrimental effect in the cell, so the unknotting process is vital to continued DNA replication. Topoisomerase IV is uniquely able to unknot DNA independently of its supercoil unwinding action, and gyrase alone can't compensate for topoisomerase IV's unknotting action. The evidence that gyrase can't perform the same actions as topoisomerase IV indicates that each topoisomerase has unique, non-overlapping actions in the cell 20)