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DNA Gyrase in Escherichia coli


DNA gyrase is a Type II topoisomerase that catalyzes the introduction of negative supercoils in DNA in the presence of ATP. DNA supercoiling refers to the overwinding (positive supercoiling) or underwinding (negative supercoiling) of the DNA strand. The amount of supercoiling in DNA is an expression of the strain felt by DNA. The introduction of these negative supercoils is necessary to relieve the strain caused by the unwinding of DNA by helicase. DNA gyrase can also introduce negative supercoils in order to remove knots and to unlink catenanes generated by recombination. By introducing these negative supercoils, DNA gyrase prevents the slow-down and eventual disruption of DNA replication. In E. coli, the introduction of these negative supercoils is essential to initiate and maintain replication fork formation.1) This article will focus on E. coli DNA gyrase and its role in the replication and transcription of plasmid DNA.


Structures of DNA gyrase

Figure 1. Structures of DNA Gyrase.2)

DNA gyrase is a heterotetramer holoenzyme seen in Figure 1. Because it is a holoenzyme, it needs to be bound to a cofactor (ATP) in order for it to be catalytically active. It consists of two A (GyrA) and B (GyrB) subunits.3)

Gyrase A

Figure 2. GyrA dimer of DNA gyrase hetertetramer.

Figure 2. GyrA dimer of DNA gyrase hetertetramer.4)

Gyrase A (GyrA) has an active center for the reactions of introducing and resealing the cuts of double-stranded DNA. The C terminus of the GyrA subunit is responsible for the unique negative supercoiling activity of DNA gyrase, and mutants lacking that C terminus lose the ability to form negative supercoils. The N terminus of the GyrA subunit is responsible for cleaving DNA via phosphodiester bonds between the 5-phosphate group of DNA and two tyrosine 122 groups, one on each GyrA subunit.5)

Gyrase B

Figure 3. GyrB dimmer of DNA gyrase heterotetramer.

Figure 3. GyrB dimmer of DNA gyrase heterotetramer.6)

Gyrase B (GyrB) contains the site of ATP hydrolysis and a number of novel features and protein folds. The N terminus of the GyrB subunit mediates its ATPase activity, and the C terminus of that subunit binds to the GyrA subunit and DNA.7) In its structure shown in Figure 3, the cavity formed by the dimer is approximately the diameter of DNA. Because of this, it was proposed that Arg286 residues, which are inside of the cavity, form some kind of binding surface for DNA in which the cavity interacts with the passage helix during strand passage. With mutation of GyrB to replace the Arg286 residue with Gln, a decrease in supercoiling activity was observed. This decrease in supercoiling activity demonstrates the importance of the Arg286 residue on strand passage of supercoiling.8)

Mechanism of DNA Supercoiling

The mechanism of DNA supercoiling by gyrase involves the following steps:9)

  1. The binding of gyrase to DNA
  2. Wrapping of a segment of DNA (∼130 bp) around the A2B2 complex
  3. Cleavage of this wrapped DNA in both strands, involving the formation of covalent bonds between the 5′-phosphates at the break sites and Tyr122 of the GyrA subunits
  4. Passage of another segment of DNA through this break
  5. Resealing of the break

In order to introduce these supercoils, which are energetically unfavorable, catalytic supercoiling requires the hydrolysis of ATP.


DNA gyrase is an essential enzyme in DNA replication in Escherichia coli. It mediates the introduction of negative supercoils near oriC, removal of positive supercoils ahead of the growing DNA fork, and separation of the two daughter duplexes. This supercoiling is essential for the initiation of DNA replication and general transcription, which requires the assistance of negative supercoiling to unwind origins and promoters. 10) As discussed previously, unwinding of the DNA duplex generates positive superhelical stress ahead of the fork that is removed by gyrase through the introduction of the negative supercoils. Both DNA replication and transcription require unwinding of long stretches of the DNA duplex in the elongation phase. Transcription itself generates positive supercoils ahead of the translocating RNA polymerase that are rapidly resolved by DNA gyrase. Gyrase introduces negative supercoils to remove this postive supercoiling and relieve the superhelical stress generated by unwinding. If gyrase is inhibited by mutation, replication slows and eventually halts when the torsional stress becomes too large.11) Because of this, the inhibition of DNA gyrase through the use of quinolines and other compounds has been the center of antimicrobial drug discovery. In order to visualize the superhelical stress caused by the unwinding of DNA by helicase, Figure 4 demonstrates the process of unwinding plasmid DNA and separating two daughter duplexes with the help of gyrase.

Figure 4. Model for DNA unlinking.

Figure 4. Model for DNA unlinking.12)

In Figure 4, stage A denotes the early stage of replication. In this stage, helicase, DNA synthesis (Syn), and associated factors have progressed only a short way from the replication origin (Ori). The unreplicated region is large, and DNA unlinking by gyrase occurs primarily in front of the fork to alleviate superhelical stress. In stage B or the Intermediate stage, the unreplicated and replicated regions are similar in size and unlinking may take place both in front of and behind the fork. In stage C or the terminal stage of replication, upon denaturation of the last few turns of the helix, these turns plus any interlinks remaining from the previous stages are converted into catenane nodes between the daughter duplexes. Catenane nodes are a topological feature that describes the interlinking and double crossover of molecules in this case DNA. These nodes are unlinked by gyrase.

From this figure, it should be noted that the unwinding of plasmid DNA results in superhelical stress. In order to remove these positive supercoils and facilitate replication, gyrase will cleave the DNA, pass one strand of DNA through the break, and reseal the break in order to reduce the strain. During replication of E. coli DNA, fork movement can generate topological changes in both the unreplicated region ahead of the fork and in the already replicated region behind the fork. Early after initiation, in stage A and B of Figure 4, movement of a replication fork causes overwinding of the DNA in the unreplicated regions, and the resulting positive supercoils are rapidly removed by DNA gyrase. As replication proceeds the overwinding in the unreplicated region can diffuse back into the already-replicated region to cause interwinding of the two daughter duplexes, as seen in stage C. These interwindings are referred to as precatenanes because if not removed, they cause the two daughter molecules to be catenated at the end of replication. These excess helical windings generated by replication can be distributed both in front of and behind the replication fork and are therefore global supercoils. Because of this, DNA gyrase is required to relax positive supercoils in front of the fork and also to “unwind” the daughter duplexes behind the replication fork in order for fork movement during replication to be maintained.

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2) Champoux, J. J. DNA Topoisomerases: Structure, Function, and Mechanism. Annu. Rev. Biochem. 2001. 70, 369–413.
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4) Saíz-Urraa, L.; Cabreraa, M. A.; Froeyen, M. Exploring the conformational changes of the ATP binding site of gyrase B from Escherichia coli complexed with different established inhibitors by using molecular dynamics simulation: Protein–ligand interactions in the light of the alanine scanning and free energy decomposition methods. J. Mol. Graphics Modell. 2011, 29, 5, 726-739.
5) , 7) Kampranis, S. C., A. D. Bates, and A. Maxwell. A model for the mechanism of strand passage by DNA gyrase. Proc. Natl. Acad. Sci. 1999, 96, 8414–8419.
6) , 9) Tingey, A. P.; Maxwell, A. Probing the role of the ATP-operated clamp in the strand-passage reaction of DNA gyrase. Nucleic Acids Research. 1996; 24 (24), 4868-4873
8) Wigley,D. B.; Davies, G. J.; Dodson, E. J.; Maxwell, A.; Dodson, G. Crystal structure of an N-terminal fragment of the DNA gyrase B protein. Nature. 1991, 351, 624–629.
10) Funnell, B.E.; Baker, T.A.; Kornberg, A. Complete enzymatic replication of plasmids containing the origin of the Escherichia coli chromosome. J. Biol. Chem.1986, 261, 5616-5624.
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chem331/dna_gyrase_in_escherichia_coli.txt · Last modified: 2016/06/07 09:53 (external edit)