DNA gyrase

DNA gyrase is a tetrameric enzyme consisting of two A (GyrA) and two B subunits (GyrB). Structurally, the complex is formed by three pairs of "gates", the sequential opening and closing of which leads to the targeted transfer of the DNA segment and the introduction of two negative super-turns. N-gates are formed by ATPase domains of B-subunits. The binding of two ATP molecules stimulates dimerization and, accordingly, the closure of the N-gate, while ATP hydrolysis to ADP , on the contrary, is the opening of the gate. DNA gates contain a catalytic center that reversibly introduces a double-stranded break in DNA, and are formed by all subunits of the enzyme. C-gates consist only of A subunits of gyrase ^[5] . A and B of the DNA gyrase subunit are homologous to proteins C and E of , as well as to the C- and N-terminal domains of eukaryotic respectively ^[6] .

At present, the mechanism of action of DNA gyrase, called the chain transfer mechanism (from the English strand passage mechanism), is considered generally accepted. According to this model, DNA gyrase interacts with two functional regions of DNA - T and G segments. At the first stage, the enzyme binds the G-segment and wraps the DNA around itself, forming a supercoil corresponding to positive supercoiling . A key role in DNA wrapping is played by the C-terminal domains of the A subunits ( CTD , from the English C-terminal domains). The addition of two ATP molecules leads to the closure of the N-gate formed by the B-subunits of the enzyme, and the binding of the T-segment of DNA. Conformational rearrangements of the complex cause the hydrolysis of the first ATP molecule and the cleavage of the G-segment due to the attack of phosphodiester nucleic acid bonds by tyrosines of the catalytic center of DNA gyrase. In the next step, the T-segment is carried through a double-stranded gap in the G-segment and the G-segment is closed back. At the final stage of the catalytic cycle, the T segment leaves the enzyme through the C-gate gyrase formed by the A subunits and the second ATP molecule is hydrolyzed ^[7] . The introduction of two negative supercoils occurs due to the inversion of the supercoil sign: a positive supercoil formed at the beginning of the catalytic cycle due to DNA wrapping around the enzyme by directed transfer of the T segment through a double stranded gap in the G segment turns into a negative supercoil ^[8] . In mathematical terms, this operation is equivalent to changing the gearing coefficient by -2. According to some estimates, the speed of gyrase reaches about 100 super-turns per second ^[9] .

It has been shown that DNA gyrase has a pronounced specificity for DNA sequences. Thus, strong binding sites of the enzyme from the bacteriophage Mu and some plasmids (pSC101, pBR322) are known. Mapping of DNA gyrase binding sites in the E. coli genome by Topo-Seq revealed a long (130 nt) binding motif explaining the existence of strong sites and reflecting the DNA wrapping around the enzymatic complex and the flexibility of the nucleic acid. An analysis of the motif revealed regions of DNA binding to the C-terminal domains of the A subunits, which are characterized by a periodic nucleotide pattern from AT and GC-rich regions with a period close to the double helix period of DNA (~ 10.5 nt) ^[3] . Previously, a similar regularity in the binding motif was found for eukaryotic nucleosomes , around which DNA also wraps (146 nt, organized in 1.8 turns) ^[10] . In total, several thousand enzyme sites were found in the E. coli genome ^[3] .

As it was shown above, gyrase has the ability to relax positive super-threads, replacing them with negative ones. This makes gyrase extremely important for cellular processes during which the DNA double strand unwinds, such as DNA replication and transcription . When DNA or RNA polymerase moves through the DNA, positive supercoils accumulate in front of the enzyme. Stress created in this way prevents the further advancement of the enzyme. This problem is solved by gyrase (as well as topoisomerase IV in the case of replication), which relaxes positive super-wraps. Thus, gyrase plays an important role both in the initiation and elongation of the processes of matrix synthesis with DNA ^[8] .

Gyrase is present in prokaryotes and some eukaryotes, however, in different species, these enzymes have different amino acid sequences and spatial structures. DNA gyrase is absent in humans, and therefore it is convenient to use it as a target for antibiotics. There are two classes of antibiotics aimed at suppressing gyrase:

• (including novobiocin). Their action is based on the principle of : they bind to the ATP-binding site of subunit B and thereby disrupt the enzyme.
• Quinolones (including nalidixic acid and ciprofloxacin). Quinolones belong to the group of “poisons” of topoisomerases. Binding to gyrase, they prevent the enzyme from conducting a G-segment DNA closure reaction, which leads to the accumulation of double-stranded breaks in nucleic acid and cell death. Topoisomerase IV is also susceptible to quinolones. Bacteria resistant to these compounds have mutant enzymes with a lower affinity for quinolones.

In addition to DNA gyrase, which induces the formation of negative supercoils, there is also reverse gyrase , which causes the formation of positive supercoils also with the expenditure of ATP hydrolysis energy. At the moment, reverse gyrase is found exclusively in hyperthermophilic archaea and bacteria, while DNA gyrase is mainly found in mesophilic bacteria. Several unique cases have been reported when both enzymes are present in one organism — the hyperthermophilic bacterium Thermotoga maritima and the hyperthermophilic archaea Archaeoglobus fulgidus ^[6] . The presence of reverse gyrase in thermophilic archaea is associated with the presence of genetic elements ( plasmids , viral DNA) in a unique positively twisted form, while the plasmids of mesophilic archaea and bacteria are negatively twisted. It is believed that positive supercoiling additionally stabilizes the DNA double helix and prevents thermal denaturation of the nucleic acid at elevated temperatures ^[11] .

Reverse gyrase is a unique combination of classical type I topoisomerase and a protein complex with helicase properties ^[6] .

• Konichev A.S., Sevastyanova G.A. Molecular biology. - Publishing Center "Academy", 2012. - 400 p. - ISBN 978-5-7695-9147-1 .