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Untangling DNA with Gyrase


By: Carol A. Rouzer, VICB Communications
Published:  October 2, 2017


New research reveals how bacterial gyrase is uniquely equipped to remove (+) DNA supercoils during replication and transcription.


In its relaxed form, B-DNA exists as a two-stranded double helix with one twist around the helical axis every 10.4-10.5 base pairs. However, most DNA in cells is present in a supercoiled state in which the double helix has been twisted to either add [(+) supercoiling] or subtract [(-) supercoiling] twists to the helix. Supercoiling induces strain in the DNA structure, leading it to adopt a new shape, often involving loops or twists. Supercoiling also strongly influences the ability of DNA processing enzymes to carry out their function. In general, mild (-) supercoiling partially unwinds the helix, increasing accessibility for transcription or replication machinery, whereas (+) supercoils create a more compact, inaccessible form. In bacteria, the circular chromosome is normally maintained with low levels of (-) supercoiling. During replication or transcription, however, unwinding of the DNA produces tight (+) supercoils ahead of the processing machinery. Bacteria have two key enzymes, gyrase and topoisomerase IV, that can unwind these supercoils, yet relatively little is known about how they recognize and interact with positively supercoiled DNA. To address this question, Vanderbilt Institute of Chemical Biology member Neil Osheroff and his doctoral student, Rachel Ashley took a close look at the interaction of both enzymes with DNA plasmids bearing equivalent degrees of (+) or (-) supercoiling. Their results provide key insights into the likely role of each enzyme during DNA replication and transcription [R. E. Ashley et al., (2017) Nuc. Acids Res., 45, 9611].


DNA gyrase and topoisomerase IV share similar structures and mechanisms of action. Both are heterotetramers comprising two each of two distinct subunits. The resultant complex contains two gates, designated N and C (Figure 1). The catalytic cycle of the enzyme begins with binding the G (or Gate) segment of DNA, which enters through the N gate. Next, the enzyme binds a second segment, the T (or Transport) segment, also via the N gate. Formation of a cleavage complex follows as the enzyme cuts the G segment, retaining the end of each strand covalently bound to a critical active site tyrosine residue in two of the subunits. Hydrolysis of two molecules of ATP provides the energy required for the enzyme to then pass the T segment of DNA through the break in the G segment and out through the C gate. Finally, the enzyme rejoins the ends of the G segment.



FIGURE 1. Structure and mechanism of DNA gyrase. (a) The enzyme is a heterotetramer of two monomers, GyrA (blue) and GyrB (yellow). The N-terminal domains of GyrA interact with DNA, and together with GyrB, form the C gate. The C-terminal domains (CTD, light blue spheres) are attached to the N-terminal domains by a flexible linker that allows them to rotate so they can bind and wrap DNA. GyrB binds and hydrolyzes ATP and forms the N-gate. (b) Mechanism for (-) supercoiling by gyrase. The gyrase first binds a segment of DNA, referred to as the G (or Gate) segment, which enters through the N gate (1). The enzyme then wraps the DNA so that a second segment (the T or Transport segment) also enters through the N gate (2). Two molecules of ATP are bound (3). Hydrolysis of ATP then drives cleavage of the G segment and passage of the T segment through the G segment and out through the C gate (4). Then, the G segment is religated (5). Image reproduced by permission from Macmillan Publishers Ltd, from M. Nöllmann, et al., (2007) Nat. Struct. Mol. Biol, 14, 264. Copyright 2007.



Despite their similarities, gyrase and topoisomerase IV exhibit key functional differences (Figure 2). In general, topoisomerase IV binds G and T segments that are at some distance from each other, or may be on different DNA molecules. It can unwind both (+) and (-) supercoils and tends to function behind the replication machinery to prevent the accumulation of precatenanes and to remove knots and tangles. In contrast, gyrase uses a wrapping mechanism that enables it to bind a T segment that is close to the G segment. It unwinds (+) but not (-) supercoils and, due to its ability to wrap DNA, it is distinguished as the only topoisomerase capable of introducing (-) supercoils. Gyrase appears to work primarily ahead of replication or transcription machinery to clear away (+) supercoils as they form and to help maintain mild (-) supercoiling during the resting state. The primary goal of the Osheroff lab was to better understand the role of each enzyme in removing (+) supercoils.

FIGURE 2. (Top) DNA structures that can form during replication. Ahead of the replication machinery (yellow oval), the DNA becomes positively supercoiled, whereas behind the machinery precatenanes form between the two new daughter strands. Topoisomerase IV can remove both kinds of structures, but likely works primarily behind the replication machinery. Gyrase is the major enzyme to remove (+) supercoils. (Bottom) Mechanistic differences between topoisomerase IV (left) and gyrase (right). Topoisomerase IV binds a T segment at some distance from the G segment, whereas gyrase uses a wrapping mechanism, enabling it to bind G and T segments close to each other. Figure reproduced under the Creative Commons Attribution License 4.0 from R. E. Ashley et al., (2017) Nuc. Acids Res., 45, 9611. Copyright 2017, Ashley, et al.



The investigators began with an agarose gel-based assay to determine the ability of the gyrase from Bacillus anthracis to unwind a positively supercoiled plasmid and then introduce negative supercoiling into that same plasmid. They found that the enzyme very rapidly unwound the (+) supercoiling, completing the job in less than 2 minutes in a highly efficient and processive manner. In contrast, the introduction of negative supercoiling occurred more slowly (requiring 30 - 45 minutes) and in a much more distributive fashion. The rates of both reactions were dependent on the concentration of ATP and exhibited similar KM values, so the striking differences could not be attributed to differences in ATP requirements. Assays of the gyrase from Escherichia coli produced very similar results.


The researchers next used magnetic tweezers to enable them to monitor the unwinding of positively supercoiled DNA at single molecule resolution. In this assay, a molecule of DNA attached at one end to a magnetic bead and at the other end to a fixed support is subjected to magnetic forces that rotate the bead, introducing a supercoil. Enzyme molecules in the surrounding solution then remove the supercoiling, which can be monitored as an increase in the length of the DNA (Figure 3). Results demonstrated that gyrase could remove 50 (+) supercoils at an average rate of > 10/s (319/492 trials) or > 20/s (110/492 trials). The reaction occurred in very rapid bursts with a mean size of 6 supercoils/burst and a rate of 107 supercoils/s interspersed with slower, steadier activity. The overall kinetics of the reaction suggested that a single molecule of gyrase completely unwound the bead-associated molecule of DNA in each trial.



FIGURE 3. Monitoring gyrase activity at single molecule resolution. (Right) A molecule of DNA is attached to a slide at one end and a magnetic bead at the other. A magnetic field exerts forces on the bead to pull it upward (black arrow) or rotate it (red arrow). (Left) Rotating the bead introduces (+) supercoils in the DNA, shortening it (red arrowhead). The action of gyrase relieves the supercoiling, allowing the DNA to return to its original length as indicated by the traces between the red arrowheads. Figure reproduced under the Creative Commons Attribution License 4.0 from R. E. Ashley et al., (2017) Nuc. Acids Res., 45, 9611. Copyright 2017, Ashley, et al.


Prior research had shown that gyrase uses its wrapping mechanism to introduce (-) supercoils in DNA, but the potential role of wrapping in removing (+) supercoils had not been investigated. To address this question, the investigators expressed the B. anthracis enzyme bearing a mutation that prevents wrapping but allows the enzyme to work by the more common strand passage mechanism observed with topoisomerase IV (Figure 2). Consistent with prior findings, this enzyme could not introduce negative supercoils into DNA. It could, however, remove both (+) and (-) supercoils. Compared to the wild-type enzyme, its rate of (+) supercoil removal was markedly slower and less processive. Removal of (-) supercoils occurred even more slowly than that of (+) supercoils, indicating that the enzyme differentiated between the two structures. These findings indicated that the extremely high efficiency of (+) supercoil removal by gyrase requires its DNA wrapping ability.


The researchers next evaluated the ability of topoisomerase IV from B. anthracis to remove both (+) and (-) supercoils. They found that the enzyme removed (+) supercoils approximately three times faster than (-) supercoils, a finding qualitatively similar to results reported previously for the enzyme from E. coli. This confirms that topoisomerase IV distinguishes between the two DNA topologies. It is also notable that the rate of (+) supercoil removal by topoisomerase IV was much slower than that of gyrase.


As noted above, an early step in the mechanism of both gyrase and topoisomerase IV is the formation of a cleavage complex. Because this step introduces a double-strand break in the DNA, it carries with it a risk of DNA damage should something interrupt normal enzyme function. The quinolone class of antibacterials binds to the cleavage complex of both topoisomerase IV and gyrase, sharply increasing levels of double-strand breaks, eventually leading to cell death. The investigators incubated gyrase from B. anthracis or E. coli with either (+) or (-) supercoiled DNA in the presence of several different quinolone antibacterials and measured the formation of DNA strand breaks. The findings indicated that both gyrase enzymes formed DNA strand breaks at much higher levels with (-) supercoiled DNA than (+) supercoiled DNA regardless of the antibacterial in the reaction mixture. This indicates that gyrase maintains higher levels of cleavage complexes with (-) than with (+) supercoiled DNA. In contrast, when the same experiment was carried out with B. anthracis topoisomerase IV, strand breaks formed at a level similar to that observed for gyrase with (-) supercoiled DNA, and little difference was observed with respect to the direction of DNA supercoiling.

Together, the findings indicate that gyrase is, by far, the most efficient enzyme for removal of (+) supercoils in DNA and that it does so with minimal accumulation of cleavage complexes. This suggests that gyrase is the best enzyme for the removal of positively DNA supercoils ahead of the replication or transcription machinery with regard to both speed and likelihood of introducing DNA damage. The exceptional capabilities of gyrase, in terms of (+) supercoil removal and (-) supercoil formation, are a direct result of its capacity to wrap DNA. The authors note that, compared to eukaryotic cells, bacteria replicate their DNA at a very high rate. It is possible that the bacterial gyrase evolved for the express purpose of rapidly disentangling DNA ahead of the fast-moving bacterial replication machinery.



View Sciences Advances article: Activities of gyrase and topoisomerase IV on positively supercoiled DNA










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