DNA gyrase

DNA gyrase (or simply gyrase) - an enzyme of the bacterium E. coli and other prokaryotes, belongs to the group of topoisomerases, which causes the formation of negative supercoils in the relaxed ring DNA molecule. Although DNA gyrase belongs to type II topoisomerases and, like them, introduces double-stranded breaks in DNA, it does this without consuming ATP energy [1].
In 2007, gyrase was described in the simplest parasitic Plasmodium falciparum of the Apicomplex type. . Unlike E. coli gyrase, it requires ATP costs [2].
Bacterial DNA gyrase is necessary for DNA replication to proceed. It is the target of many antibiotics, for example, nalidixic acid, novobiocin [en] and ciprofloxacin.
DNA gyrase was described in M. Gellert et al. In 1976 [3].
Contents
1 Mechanism
2 Biological role
3 Interaction with antibiotics
4 Reverse gyrase
5 Notes
6 Literature
Mechanism
The mechanism of formation of negative supercoils of DNA-gyrase
In general, the mechanism of action of DNA -girazy is that. It interacts with DNA so that DNA is wound around it. In those places of DNA that are associated with gyrase, positive supercoiling occurs. After this, the enzyme introduces a double-stranded break in the DNA and transfers the double strand from the inside to the outside. After this, gyrase sews both breaks, thus turning positive supercoils into negative ones [1]. The speed of gyrase reaches about 100 supercoils per minute [3].
The above model of the action of DNA gyrase is confirmed by a number of experimental data. It is known that around a enzyme about 120 N in length is wrapped around the enzyme. n. More precisely, gyrase is a tetramer, and DNA combines with the enzyme at two sites. It was also found that the interaction of DNA and the enzyme is not limited to double-stranded breakage: the 5'-end of the DNA binds to the 122 amino acid residue (tyrosine) of the gyrase (subunit A, see below), and the 3'-OH end remains free. Since the binding of DNA and gyrase is specific, 2 specific sites with the following consensus sequence were found in E. coli DNA: 5'-RNNNRNNRTGRYCTYNYNGNY-3 '[4].
It was found that the gap occurs between TG nucleotides ( highlighted above). The entire genome of E. coli presumably has about 45-50 large DNA-gyrase binding sites, but this does not mean that they are all functional. There are also almost 10 thousand sites where there is weak DNA and gyrase binding. This means that large sites account for approximately 0.5% of all possible binding sites. However, even these sites in the vast majority of cases do not show any activity and work only when the gyrase is in extremely high concentration [4].
It is known that for the formation of two negative supercoils (that is, for the implementation of one catalytic cycle) 2 ATP molecules are needed . However, experiments on the operation of gyrase in the presence and absence of ATP gave unexpected results. As expected, the presence of ATP promotes the formation of negative DNA supercoils, but in the absence of ATP, negative supercoils unwound. From this it follows that the function of gyrase for introducing double-stranded breaks is neither activated nor is dependent on the presence of ATP [4]. However, ATP is still necessary for the normal operation of gyrase.
E. coli gyrase consists of two subunits: A (GyrA) and B (GyrB). They are homologous to the C and E proteins of topoisomerase IV [en], as well as to the C- and N-terminal domains of the eukaryotic topoisomerase II [en], respectively [5]. It was shown that subunit B has a very weak ATPase activity, which is very strongly stimulated by the presence of a substrate (i.e., ATP), DNA, and subunit A. Subunit A introduces a double-stranded gap (ATP is not needed for this process), subunit B carries one of the chains ( this is done at the expense of ATP), and then subunit A cross-links the chains. It was experimentally established that enzyme activation occurs only when both of its subunits bind to DNA [4].
Biological role
As shown above, gyrase has the ability to relax positive supercoils, replacing them with negative ones. This makes gyrase extremely important for the process of DNA replication. When DNA polymerase moves through the DNA, positive supercoils form in front of the enzyme, as the DNA helix is twisted right. Stress created in this way prevents the further advancement of DNA polymerase. This problem is solved by gyrase (as well as topoisomerase IV), which relaxes positive super-wakes. Thus, gyrase plays an important role both in the initiation and in the elongation and termination of replication [1].
Interaction with antibiotics
DNA gyrase in combination with DNA and two ciprofloxacin molecules
Gyrase is present in prokaryotes and of 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: Aminocoumarins [en] (including novobiocin). Their action is based on the principle of competitive inhibition: they bind to the ATP-binding site of subunit B and thereby disrupt the enzyme.
Quinolones (including nalidixic acid and ciprofloxacin). Quinolones bind to gyrase, preventing it from taking off positive super-threads and thus inhibiting DNA replication. Quinolone-resistant bacteria have mutant topoisomerases that do not bind to quinolones.
Reverse gyrase
In addition to DNA gyrase, which induces the formation of negative supercoils, there is also a reverse gyrase that causes the formation of positive supercoils. At the moment, reverse gyrase is found in all hyperthermophilic organisms, while DNA gyrase is found only in mesophiles. Reverse gyrase is present in both bacteria and archaea, however, DNA gyrase is found only in bacteria, although halophilic archaea have identified genes presumably encoding DNA gyrase. 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 plasmids of mesophilic archaea and bacteria are negatively twisted [5].
Reverse gyrase is a unique combination of classical type I topoisomerases and a protein complex with helicase properties. DNA gyrase, by contrast, belongs to the classical family of type II topoisomerases, but has the unique ability to form a positive toroidal supercoil in the DNA segment [5].
Notes
↑ 1 2 3 Konichev, Sevastyanova, 2012, p. 100.
↑ Mohd Ashraf Dar, Atul Sharma, Neelima Mondal, Suman Kumar Dhar Molecular Cloning of Apicoplast-Targeted Plasmodium falciparum DNA Gyrase Genes: Unique Intrinsic ATPase Activity and ATP-Independent Dimerization of PfGyrB Subunit // Euk 2007. - T. 6, No. 3. - S. 398-412. - DOI: 10.1128 / EC.00357-06.
↑ 1 2 Molecular Biology and Genetics. Explanatory Dictionary: DNA gyrase.
↑ 1 2 3 4 Mechanism of DNA Gyrase.
↑ 1 2 3 Guipaud O., Marguet E., Noll KM, de la Tour CB, Forterre P. Both DNA gyrase and reverse gyrase are present in the hyperthermophilic bacterium Thermotoga maritima. // Proc Natl Acad Sci USA .. - 1997. - T. 94, No. 20. - P. 10606-11.
Literature
Konichev A. S., Sevastyanova G. A. Molecular Biology. - Publishing Center "Academy", 2012. - 400 p. - ISBN 978-5-7695-9147-1. - DNA replication - Initiation - Prokaryotes - Prereplication complex
Helicases (DnaA [en] • DnaB • T7 [en])
Primase (DnaG)
Eukaryotes [en]
Prereplication complex
Recognition complex Ori [en] (ORC1 [en] • ORC2 [en] • ORC3 [en] • ORC4 [en] • ORC5 [en] • ORC6 [en]) • Cdc6 [en] • Cdt1 [en]
Minichromosome Service Complex [MCM2 [en] • MCM3 [en] • MCM4 [en] • MCM5 [en] • MCM6 • MCM7 [ en])
Resolving factor [en]
Autonomously replicating sequence
Proteins that bind single-stranded DNA (SSBP2 [en] • SSBP3 [en] • SSBP4) • RNase H [en] (RNASEH1 [en] • RNASEH2A [en])
Helicases: Mcm2-7
Primase: DNA gender imerase α - Common for pro- and eukaryotes - Replication origin / Replicon
Replication fork (lagging and leading chains) • Okazaki fragments • Primer
Elongation - Prokaryotes
DNA polymerase holoenzyme III (dnaC • dnaE [en] • dnaH [en] • dnaN [en] • dnaQ [en] • dnaT [en] • dnaX [en] • holA [en] • holB [en] • holC [en] • holD [en] • holE [en]) • replicoma [en] • ligase • Sliding clasp proteins • Topoisomerase (DNA gyrase)
DNA polymerase I [en] (Klenov fragment)
Eukaryotes
Replication factor C [en] RFC1 • Oscillating endonuclease [en] (FEN1 [en]) • Topoisomerase • Replicative white A (RPA1 [en])
DNA polymerase δ [en] (POLD1 [en] • POLD2 [en] • POLD3 [en] • POLD4 [en]) Sliding zipper proteins (PCNA [en])
Common to pro- and eukaryotes - Processivity • ligase - Termination - Telomeres: telomerase (TERT [en] • TERC [en] • DKC1 [en])
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