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https://proteopedia.org/api.php?action=feedcontributions&feedformat=atom&user=Laurent+Thomas Proteopedia - User contributions [en] 2026-09-27T18:13:22Z User contributions MediaWiki 1.43.8 https://proteopedia.org/index.php?title=User:Laurent_Thomas/Sandbox_1&diff=1882674 User:Laurent Thomas/Sandbox 1 2014-01-04T13:22:32Z

<p>Laurent Thomas: </p> <hr /> <div>This is my own sandbox !<br /> {{STRUCTURE_1ab4| PDB=1ab4 | CAPTION='hello'| SCENE=56/568026/Coloured/1}}</div>

Laurent Thomas https://proteopedia.org/index.php?title=Sandbox_Reserved_828&diff=1882672 Sandbox Reserved 828 2014-01-04T13:22:14Z

<p>Laurent Thomas: /* '''Introduction :''' */</p> <hr /> <div><br /> {{Sandbox_Reserved_ESBS}}<br /> <br /> <br /> <br /> ==E.Coli Gyrase==<br /> ----<br /> {{STRUCTURE_1ab4| PDB=1ab4 | CAPTION=A-subunit (monomer), resolution 2.80Å, 1ab4.pdb| SCENE=56/568026/Coloured/1}}<br /> [[Image:1ab4.png|left|200px]]<br /> <br /> ==='''Introduction'''===<br /> <br /> <br /> Gyrase is the '''only prokaryote DNA topoisomerase II able to introduce negative supercoils in the DNA''' (see [http://en.wikipedia.org/wiki/DNA_supercoil DNA Supercoil]) in order to remove positive supercoils. It '''catalyses the hydrolysis of two phosphodiester bonds in a DNA segment (DNA cleavage)''' called G segment. Then, thanks to '''ATP dependant conformation changes''', it enables the passage of another segment (the T segment) through the break, and then religates the broken segment. Gyrase acts prior to the replication (before the replication fork) or other mecanisms requiring loose DNA. <br /> '''In abscence of ATP, like other topoisomerases II, gyrase only relaxes supercoils.'''<br /> <br /> Gyrase is '''coded by two differents contiguous genes gyrA and gyrB''' as it is a 350 kDa '''A<sub>2</sub>B<sub>2</sub> heterotetramers''' of two A proteins and two B proteins. <br /> <br /> The A protein breaks and religates DNA. <br /> The B protein has ATPase activity.<br /> <br /> ----<br /> <br /> ==='''Structure'''===<br /> <br /> [[Image:Gyrasegene.jpg]]<br /> <br /> '''The A protein''' breaks and religates DNA as the DNA cleavage core and the CTD lies on it. The smallest fragment capable of catalyzing DNA breakage and reunion is between residues 7 and 523 of A sequence, it is the gyrA59 (59kDa). <br /> The gyrA N-ter domain is called '''breakage and reunion domain'''. It is composed of two domains at the <scene name='56/568026/Head/2'>head</scene> region :<br /> <br /> *a winged-helix-turn-helix domain or <scene name='56/568026/Whd/1'>winged helix domain (WHD</scene>) where lies the catalytic tyrosines: the active-site tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> are on loops at either end of the dimer interface.<br /> <br /> *a <scene name='56/568026/Tower/1'>tower domain</scene> with alpha/beta structure that participates in DNA bending during cleavage.<br /> <br /> The '''‘head’ dimer interface is dominated by an antiparallel side by-side packing of the <scene name='56/568026/Alpha3/2'>alpha 3</scene> (the first helix of the HTH motif) from each monomer''', together with their adjoining loops. At the top of the interface, '''the ‘recognition’ helices <scene name='56/568026/Alpha4/1'>alpha 4</scene> make a head-to-head antiparallel dimer contact.'''<br /> <br /> <br /> There is a '''<scene name='56/568026/Coiledcoil_141819/1'>Coiled-coil</scene> domain''', folowing the tower, with a helical core at the tail region. Two long helices (<br /> <scene name='56/568026/Helix14/1'>alpha helix 14</scene> and <scene name='56/568026/Helix18/1'>alpha helix 18</scene>) emanate from this core and connect, together with the C-terminal helix (<scene name='56/568026/Helix19/1'>alpha helix 19</scene>), the head and tail fragments. This domain has a small globular domain at it's end and is '''involved in the dimerization creating the <scene name='56/568026/Coiledcoil/1'>C-gate</scene>.'''<br /> <br /> <br /> The '''three connecting helices (<scene name='56/568026/Helix14/1'>α14</scene>, <scene name='56/568026/Helix18/1'>α18</scene> and <scene name='56/568026/Helix19/1'>α19</scene>)''' adopt very different conformations, leading to '''large quaternary movements''' involving a single hinge-point within the helices and '''rigid body movements of the head fragments.''' <br /> <br /> [[Image:helixrot.jpg]] [[Image:gyrA592.jpg]]<br /> <br /> The tail is structurally conserved although large surface loops emanating from different points give it a different outward appearance. <br /> '''GyrA forms a heart-shaped homodimer with two protein interfaces, the DNA- and C-gates'''. GyrA59 is the minimal fragment of the A-subunit which, when complexed with the B-subunit, has DNA-cleavage activity. <br /> The remaining 30–35 kDa comprising the C-terminal domain (CTD) of GyrA shows a domain forming a b-pinwheel with a positively charged amino-acid perimeter. This carboxy-terminal domain of GyrA (cyan) is required for the introduction of DNA supercoils), DNA wraps around it. It is linked to the central part of Gyrase A by a flexible 13-15 residues region and stands close to the C-gate and may present an up and down movement in the early stades of the catalytic cycle and may be involved in the helping of G segment and D-gate binding so as the CTD binding of DNA.<br /> <br /> '''The B protein''' has the GHKL ATPase domain, a transducer domain and the Toprim fold on it. <br /> Two ATPase domains from two monomers dimerize to form a closed conformation, the N-gate. <br /> The Toprim fold is a Rossmann fold (beta-alpha-beta-alpha-beta) that contains three invariant acidic residues that coordinate magnesium ions involved in DNA cleavage and DNA religation. This torpim fold interacts with the Winged helix domain of gyrA to create the D-gate.<br /> <br /> <br /> '''The central core''' of the protein is made out of the association of GyrA and Gyr B with the Toprim fold, the DNA-binding domain (WHD)and tower domain, all three creating the D-gate.<br /> These domains are composed of numerous positively charged residues to interact with DNA.<br /> The Toprim domain is flexible and that this flexibility can allow the Toprim domain to coordinate with <br /> the WHD to form a competent cleavage complex.<br /> DNA is bent by ~150 degrees through an invariant isoleucine (I172)<br /> <br /> <br /> [[Image:Gyrasegates.jpg]]<br /> <br /> <br /> ----<br /> ==='''Mechanism'''===<br /> <br /> 40-bp of duplex DNA, the G-segment, bind to the core of the enzyme and are cleaved by the active site tyrosines, while another DNA duplex, the T-segment, is captured through the ATP-induced dimerization of a protein gate, the N-gate. After passage through the transiently broken G-segment (DNA gate), the T-segment exits the protein through another protein gate, the C-gate. ATP hydrolysis and release reset the conformation of the enzyme and DNA to their initial state, poised for another strand-passage event or release of the DNA.<br /> N- and C-gates that open or close to allow T-segment transport through both the protein and the cleaved G-segment.<br /> <br /> <br /> [[Image:mecanismgyrase.jpg]]<br /> <br /> ----<br /> <br /> ==='''Reaction'''===<br /> <br /> The process of DNA supercoiling by gyrase is the following :<br /> # Gyrase binds DNA (gyrase binds more strongly to relaxed or linear DNA than to supercoiled one by a factor of about ten)<br /> # Cleavage of the G-segment<br /> # Passage of T-segment of the other strand through the cleavage site (thanks to ATP)<br /> # Reunion of the G segment<br /> # Translocation<br /> <br /> The '''A subunit is responsible for the breakage reunion of DNA whereas the B-subunit has the ATPase activity.'''<br /> '''The introduction of negative supercoils requires the energy of ATP-hydrolysis. 2 ATP are hydrolyzed per reaction (2 B-subunits in the gyrase)''' so that the linking number (Lk) changes in step of 2. The '''ATPase activity of the B subunit is partially located in the N-terminal region of protein B whereas the C-terminal is involved in the interaction with the A subunit and DNA.''' <br /> '''The B subunit has a weak ATPase activity in the absence of A and DNA, eventhought the A subunit is able to bind DNA without the B subunit. Both subunits are required for all the reactions of gyrase.'''<br /> The DNA supercoiling reaction '''requires in addition to ATP, a divalent cation such as Mg<sup>2+</sup>''', and is stimulated in the presence of spermidine. <br /> <br /> Therefore, the gyrase could also catalyse the following reaction :<br /> * Relaxation of negative/positive supercoils<br /> * Catenation/Decatenation of closed-circular DNA<br /> * DNA cleavage (induced by Quinolone)<br /> * Unknotting DNA strands<br /> <br /> Contrary to the introduction of negative supercoils, '''some of these reactions don't necessarily need the energy from ATP-hydrolysis.'''<br /> <br /> The cleavage of DNA is achieved by a '''transesterification reaction between the tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> and the target phosphoryl groups on opposing strands of the DNA backbone''', resulting in the tyrosine being covalentlty attached to the 5' end of the cleaved segment with a 4-base overhang.<br /> The '''active site tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene>''' are on loops at either end of the dimer interface, 30Å apart, and sit at the ends of strongly basic grooves created by the dimer-related monomers.<br /> <br /> The gyrase structure reveals a '''new cluster of conserved residues''', juxtaposing Tyr 122 and Arg 121 from one monomer and His 80, Arg 32 and Lys 42 from the other monomer. '''This cluster may form the active site of the breakage–reunion reaction''', with the other conserved positive charges (Arg 46 and Arg 47) anchoring the non-covalently bound 3' end of the cleaved DNA.<br /> <br /> The gyrase could be inhibited by both the [http://en.wikipedia.org/wiki/Quinolone quinolones] and [http://en.wikipedia.org/wiki/Coumarin coumarins].<br /> <br /> '''Coumarins prevent the B-subunit of the gyrase from hydrolyzing the ATP''', so that the gyrase can't come back to its starting state for another round of supercoiling. The mecanism is not yet well known.<br /> <br /> '''Quinolones''' inhibit DNA supercoiling by gyrase, it has even the ability to '''induce DNA cleavage''' instead. It appears that '''these drugs will arrest any gyrase rection involving double-strand DNA breakage.'''<br /> However, some mutations offers quinolone resistance, most of them involved a part of the GyrA sequence : between the amino acids 67 and 106. <br /> There are also others antibiotics, like the Cinodine which binds DNA and inhibits its supercoiling by gyrase.<br /> <br /> ===See Also===<br /> *[[Gyrase|Gyrase]]<br /> *[[Topoisomerase|Topoisomerase]]<br /> <br /> ===References===<br /> <ref group="xtra">PMID:009278055</ref><references group="xtra"/><br /> [[Category: Escherichia coli]]<br /> [[Category: Cabral, J H.M.]]<br /> [[Category: Liddington, R C.]]<br /> [[Category: Maxwell, A.]]<br /> [[Category: Gyrase]]<br /> [[Category: Supercoiling dna]]<br /> [[Category: Topoisomerase]]<br /> [[Category: Topoisomerase ii]]<br /> *Reece, Richard J., Anthony Maxwell, and James C. Wang. "DNA gyrase: structure and function." Critical reviews in biochemistry and molecular biology 26.3-4 (1991): 335-375.</div>

Laurent Thomas https://proteopedia.org/index.php?title=Sandbox_Reserved_828&diff=1882671 Sandbox Reserved 828 2014-01-04T13:20:37Z

<p>Laurent Thomas: </p> <hr /> <div><br /> {{Sandbox_Reserved_ESBS}}<br /> <br /> <br /> <br /> ==E.Coli Gyrase==<br /> ----<br /> {{STRUCTURE_1ab4| PDB=1ab4 | CAPTION=A-subunit (monomer), resolution 2.80Å, 1ab4.pdb| SCENE=56/568026/Coloured/1}}<br /> [[Image:1ab4.png|left|200px]]<br /> <br /> ==='''Introduction :'''===<br /> <br /> <br /> Gyrase is the '''only prokaryote DNA topoisomerase II able to introduce negative supercoils in the DNA''' (see [http://en.wikipedia.org/wiki/DNA_supercoil DNA Supercoil]) in order to remove positive supercoils. It '''catalyses the hydrolysis of two phosphodiester bonds in a DNA segment (DNA cleavage)''' called G segment. Then, thanks to '''ATP dependant conformation changes''', it enables the passage of another segment (the T segment) through the break, and then religates the broken segment. Gyrase acts prior to the replication (before the replication fork) or other mecanisms requiring loose DNA. <br /> '''In abscence of ATP, like other topoisomerases II, gyrase only relaxes supercoils.'''<br /> <br /> Gyrase is '''coded by two differents contiguous genes gyrA and gyrB''' as it is a 350 kDa '''A<sub>2</sub>B<sub>2</sub> heterotetramers''' of two A proteins and two B proteins. <br /> <br /> The A protein breaks and religates DNA. <br /> The B protein has ATPase activity.<br /> <br /> ----<br /> ==='''Structure'''===<br /> <br /> [[Image:Gyrasegene.jpg]]<br /> <br /> '''The A protein''' breaks and religates DNA as the DNA cleavage core and the CTD lies on it. The smallest fragment capable of catalyzing DNA breakage and reunion is between residues 7 and 523 of A sequence, it is the gyrA59 (59kDa). <br /> The gyrA N-ter domain is called '''breakage and reunion domain'''. It is composed of two domains at the <scene name='56/568026/Head/2'>head</scene> region :<br /> <br /> *a winged-helix-turn-helix domain or <scene name='56/568026/Whd/1'>winged helix domain (WHD</scene>) where lies the catalytic tyrosines: the active-site tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> are on loops at either end of the dimer interface.<br /> <br /> *a <scene name='56/568026/Tower/1'>tower domain</scene> with alpha/beta structure that participates in DNA bending during cleavage.<br /> <br /> The '''‘head’ dimer interface is dominated by an antiparallel side by-side packing of the <scene name='56/568026/Alpha3/2'>alpha 3</scene> (the first helix of the HTH motif) from each monomer''', together with their adjoining loops. At the top of the interface, '''the ‘recognition’ helices <scene name='56/568026/Alpha4/1'>alpha 4</scene> make a head-to-head antiparallel dimer contact.'''<br /> <br /> <br /> There is a '''<scene name='56/568026/Coiledcoil_141819/1'>Coiled-coil</scene> domain''', folowing the tower, with a helical core at the tail region. Two long helices (<br /> <scene name='56/568026/Helix14/1'>alpha helix 14</scene> and <scene name='56/568026/Helix18/1'>alpha helix 18</scene>) emanate from this core and connect, together with the C-terminal helix (<scene name='56/568026/Helix19/1'>alpha helix 19</scene>), the head and tail fragments. This domain has a small globular domain at it's end and is '''involved in the dimerization creating the <scene name='56/568026/Coiledcoil/1'>C-gate</scene>.'''<br /> <br /> <br /> The '''three connecting helices (<scene name='56/568026/Helix14/1'>α14</scene>, <scene name='56/568026/Helix18/1'>α18</scene> and <scene name='56/568026/Helix19/1'>α19</scene>)''' adopt very different conformations, leading to '''large quaternary movements''' involving a single hinge-point within the helices and '''rigid body movements of the head fragments.''' <br /> <br /> [[Image:helixrot.jpg]] [[Image:gyrA592.jpg]]<br /> <br /> The tail is structurally conserved although large surface loops emanating from different points give it a different outward appearance. <br /> '''GyrA forms a heart-shaped homodimer with two protein interfaces, the DNA- and C-gates'''. GyrA59 is the minimal fragment of the A-subunit which, when complexed with the B-subunit, has DNA-cleavage activity. <br /> The remaining 30–35 kDa comprising the C-terminal domain (CTD) of GyrA shows a domain forming a b-pinwheel with a positively charged amino-acid perimeter. This carboxy-terminal domain of GyrA (cyan) is required for the introduction of DNA supercoils), DNA wraps around it. It is linked to the central part of Gyrase A by a flexible 13-15 residues region and stands close to the C-gate and may present an up and down movement in the early stades of the catalytic cycle and may be involved in the helping of G segment and D-gate binding so as the CTD binding of DNA.<br /> <br /> '''The B protein''' has the GHKL ATPase domain, a transducer domain and the Toprim fold on it. <br /> Two ATPase domains from two monomers dimerize to form a closed conformation, the N-gate. <br /> The Toprim fold is a Rossmann fold (beta-alpha-beta-alpha-beta) that contains three invariant acidic residues that coordinate magnesium ions involved in DNA cleavage and DNA religation. This torpim fold interacts with the Winged helix domain of gyrA to create the D-gate.<br /> <br /> <br /> '''The central core''' of the protein is made out of the association of GyrA and Gyr B with the Toprim fold, the DNA-binding domain (WHD)and tower domain, all three creating the D-gate.<br /> These domains are composed of numerous positively charged residues to interact with DNA.<br /> The Toprim domain is flexible and that this flexibility can allow the Toprim domain to coordinate with <br /> the WHD to form a competent cleavage complex.<br /> DNA is bent by ~150 degrees through an invariant isoleucine (I172)<br /> <br /> <br /> [[Image:Gyrasegates.jpg]]<br /> <br /> <br /> ----<br /> ==='''Mechanism'''===<br /> <br /> 40-bp of duplex DNA, the G-segment, bind to the core of the enzyme and are cleaved by the active site tyrosines, while another DNA duplex, the T-segment, is captured through the ATP-induced dimerization of a protein gate, the N-gate. After passage through the transiently broken G-segment (DNA gate), the T-segment exits the protein through another protein gate, the C-gate. ATP hydrolysis and release reset the conformation of the enzyme and DNA to their initial state, poised for another strand-passage event or release of the DNA.<br /> N- and C-gates that open or close to allow T-segment transport through both the protein and the cleaved G-segment.<br /> <br /> <br /> [[Image:mecanismgyrase.jpg]]<br /> <br /> ----<br /> <br /> ==='''Reaction'''===<br /> <br /> The process of DNA supercoiling by gyrase is the following :<br /> # Gyrase binds DNA (gyrase binds more strongly to relaxed or linear DNA than to supercoiled one by a factor of about ten)<br /> # Cleavage of the G-segment<br /> # Passage of T-segment of the other strand through the cleavage site (thanks to ATP)<br /> # Reunion of the G segment<br /> # Translocation<br /> <br /> The '''A subunit is responsible for the breakage reunion of DNA whereas the B-subunit has the ATPase activity.'''<br /> '''The introduction of negative supercoils requires the energy of ATP-hydrolysis. 2 ATP are hydrolyzed per reaction (2 B-subunits in the gyrase)''' so that the linking number (Lk) changes in step of 2. The '''ATPase activity of the B subunit is partially located in the N-terminal region of protein B whereas the C-terminal is involved in the interaction with the A subunit and DNA.''' <br /> '''The B subunit has a weak ATPase activity in the absence of A and DNA, eventhought the A subunit is able to bind DNA without the B subunit. Both subunits are required for all the reactions of gyrase.'''<br /> The DNA supercoiling reaction '''requires in addition to ATP, a divalent cation such as Mg<sup>2+</sup>''', and is stimulated in the presence of spermidine. <br /> <br /> Therefore, the gyrase could also catalyse the following reaction :<br /> * Relaxation of negative/positive supercoils<br /> * Catenation/Decatenation of closed-circular DNA<br /> * DNA cleavage (induced by Quinolone)<br /> * Unknotting DNA strands<br /> <br /> Contrary to the introduction of negative supercoils, '''some of these reactions don't necessarily need the energy from ATP-hydrolysis.'''<br /> <br /> The cleavage of DNA is achieved by a '''transesterification reaction between the tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> and the target phosphoryl groups on opposing strands of the DNA backbone''', resulting in the tyrosine being covalentlty attached to the 5' end of the cleaved segment with a 4-base overhang.<br /> The '''active site tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene>''' are on loops at either end of the dimer interface, 30Å apart, and sit at the ends of strongly basic grooves created by the dimer-related monomers.<br /> <br /> The gyrase structure reveals a '''new cluster of conserved residues''', juxtaposing Tyr 122 and Arg 121 from one monomer and His 80, Arg 32 and Lys 42 from the other monomer. '''This cluster may form the active site of the breakage–reunion reaction''', with the other conserved positive charges (Arg 46 and Arg 47) anchoring the non-covalently bound 3' end of the cleaved DNA.<br /> <br /> The gyrase could be inhibited by both the [http://en.wikipedia.org/wiki/Quinolone quinolones] and [http://en.wikipedia.org/wiki/Coumarin coumarins].<br /> <br /> '''Coumarins prevent the B-subunit of the gyrase from hydrolyzing the ATP''', so that the gyrase can't come back to its starting state for another round of supercoiling. The mecanism is not yet well known.<br /> <br /> '''Quinolones''' inhibit DNA supercoiling by gyrase, it has even the ability to '''induce DNA cleavage''' instead. It appears that '''these drugs will arrest any gyrase rection involving double-strand DNA breakage.'''<br /> However, some mutations offers quinolone resistance, most of them involved a part of the GyrA sequence : between the amino acids 67 and 106. <br /> There are also others antibiotics, like the Cinodine which binds DNA and inhibits its supercoiling by gyrase.<br /> <br /> ===See Also===<br /> *[[Gyrase|Gyrase]]<br /> *[[Topoisomerase|Topoisomerase]]<br /> <br /> ===References===<br /> <ref group="xtra">PMID:009278055</ref><references group="xtra"/><br /> [[Category: Escherichia coli]]<br /> [[Category: Cabral, J H.M.]]<br /> [[Category: Liddington, R C.]]<br /> [[Category: Maxwell, A.]]<br /> [[Category: Gyrase]]<br /> [[Category: Supercoiling dna]]<br /> [[Category: Topoisomerase]]<br /> [[Category: Topoisomerase ii]]<br /> *Reece, Richard J., Anthony Maxwell, and James C. Wang. "DNA gyrase: structure and function." Critical reviews in biochemistry and molecular biology 26.3-4 (1991): 335-375.</div>

Laurent Thomas https://proteopedia.org/index.php?title=Sandbox_Reserved_828&diff=1882670 Sandbox Reserved 828 2014-01-04T13:19:40Z

<p>Laurent Thomas: </p> <hr /> <div><br /> {{Sandbox_Reserved_ESBS}}<br /> <br /> <br /> <br /> ==E.Coli Gyrase==<br /> ----<br /> {{STRUCTURE_1ab4| PDB=1ab4 | CAPTION=A-subunit (monomer), resolution 2.80Å, 1ab4.pdb| SCENE=56/568026/Coloured/1}}<br /> [[Image:1ab4.png|left|200px]]<br /> <br /> ==='''Introduction :'''===<br /> <br /> <br /> Gyrase is the '''only prokaryote DNA topoisomerase II able to introduce negative supercoils in the DNA''' (see [http://en.wikipedia.org/wiki/DNA_supercoil DNA Supercoil]) in order to remove positive supercoils. It '''catalyses the hydrolysis of two phosphodiester bonds in a DNA segment (DNA cleavage)''' called G segment. Then, thanks to '''ATP dependant conformation changes''', it enables the passage of another segment (the T segment) through the break, and then religates the broken segment. Gyrase acts prior to the replication (before the replication fork) or other mecanisms requiring loose DNA. <br /> '''In abscence of ATP, like other topoisomerases II, gyrase only relaxes supercoils.'''<br /> <br /> Gyrase is '''coded by two differents contiguous genes gyrA and gyrB''' as it is a 350 kDa '''A<sub>2</sub>B<sub>2</sub> heterotetramers''' of two A proteins and two B proteins. <br /> <br /> The A protein breaks and religates DNA. <br /> The B protein has ATPase activity.<br /> <br /> ----<br /> ==='''Structure'''===<br /> <br /> [[Image:Gyrasegene.jpg]]<br /> <br /> '''The A protein''' breaks and religates DNA as the DNA cleavage core and the CTD lies on it. The smallest fragment capable of catalyzing DNA breakage and reunion is between residues 7 and 523 of A sequence, it is the gyrA59 (59kDa). <br /> The gyrA N-ter domain is called '''breakage and reunion domain'''. It is composed of two domains at the <scene name='56/568026/Head/2'>head</scene> region :<br /> <br /> *a winged-helix-turn-helix domain or <scene name='56/568026/Whd/1'>winged helix domain (WHD</scene>) where lies the catalytic tyrosines: the active-site tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> are on loops at either end of the dimer interface.<br /> <br /> *a <scene name='56/568026/Tower/1'>tower domain</scene> with alpha/beta structure that participates in DNA bending during cleavage.<br /> <br /> The '''‘head’ dimer interface is dominated by an antiparallel side by-side packing of the <scene name='56/568026/Alpha3/2'>alpha 3</scene> (the first helix of the HTH motif) from each monomer''', together with their adjoining loops. At the top of the interface, '''the ‘recognition’ helices <scene name='56/568026/Alpha4/1'>alpha 4</scene> make a head-to-head antiparallel dimer contact.'''<br /> <br /> <br /> There is a '''<scene name='56/568026/Coiledcoil_141819/1'>Coiled-coil</scene> domain''', folowing the tower, with a helical core at the tail region. Two long helices (<br /> <scene name='56/568026/Helix14/1'>alpha helix 14</scene> and <scene name='56/568026/Helix18/1'>alpha helix 18</scene>) emanate from this core and connect, together with the C-terminal helix (<br /> <scene name='56/568026/Helix19/1'>alpha helix 19</scene>), the head and tail fragments. This domain has a small globular domain at it's end and is '''involved in the dimerization creating the <scene name='56/568026/Coiledcoil/1'>C-gate</scene>.'''<br /> <br /> <br /> The '''three connecting helices (<scene name='56/568026/Helix14/1'>α14</scene>, <scene name='56/568026/Helix18/1'>α18</scene> and <scene name='56/568026/Helix19/1'>α19</scene>)''' adopt very different conformations, leading to '''large quaternary movements''' involving a single hinge-point within the helices and '''rigid body movements of the head fragments.''' <br /> <br /> [[Image:helixrot.jpg]] [[Image:gyrA592.jpg]]<br /> <br /> The tail is structurally conserved although large surface loops emanating from different points give it a different outward appearance. <br /> '''GyrA forms a heart-shaped homodimer with two protein interfaces, the DNA- and C-gates'''. GyrA59 is the minimal fragment of the A-subunit which, when complexed with the B-subunit, has DNA-cleavage activity. <br /> The remaining 30–35 kDa comprising the C-terminal domain (CTD) of GyrA shows a domain forming a b-pinwheel with a positively charged amino-acid perimeter. This carboxy-terminal domain of GyrA (cyan) is required for the introduction of DNA supercoils), DNA wraps around it. It is linked to the central part of Gyrase A by a flexible 13-15 residues region and stands close to the C-gate and may present an up and down movement in the early stades of the catalytic cycle and may be involved in the helping of G segment and D-gate binding so as the CTD binding of DNA.<br /> <br /> '''The B protein''' has the GHKL ATPase domain, a transducer domain and the Toprim fold on it. <br /> Two ATPase domains from two monomers dimerize to form a closed conformation, the N-gate. <br /> The Toprim fold is a Rossmann fold (beta-alpha-beta-alpha-beta) that contains three invariant acidic residues that coordinate magnesium ions involved in DNA cleavage and DNA religation. This torpim fold interacts with the Winged helix domain of gyrA to create the D-gate.<br /> <br /> <br /> '''The central core''' of the protein is made out of the association of GyrA and Gyr B with the Toprim fold, the DNA-binding domain (WHD)and tower domain, all three creating the D-gate.<br /> These domains are composed of numerous positively charged residues to interact with DNA.<br /> The Toprim domain is flexible and that this flexibility can allow the Toprim domain to coordinate with <br /> the WHD to form a competent cleavage complex.<br /> DNA is bent by ~150 degrees through an invariant isoleucine (I172)<br /> <br /> <br /> [[Image:Gyrasegates.jpg]]<br /> <br /> <br /> ----<br /> ==='''Mechanism'''===<br /> <br /> 40-bp of duplex DNA, the G-segment, bind to the core of the enzyme and are cleaved by the active site tyrosines, while another DNA duplex, the T-segment, is captured through the ATP-induced dimerization of a protein gate, the N-gate. After passage through the transiently broken G-segment (DNA gate), the T-segment exits the protein through another protein gate, the C-gate. ATP hydrolysis and release reset the conformation of the enzyme and DNA to their initial state, poised for another strand-passage event or release of the DNA.<br /> N- and C-gates that open or close to allow T-segment transport through both the protein and the cleaved G-segment.<br /> <br /> <br /> [[Image:mecanismgyrase.jpg]]<br /> <br /> ----<br /> <br /> ==='''Reaction'''===<br /> <br /> The process of DNA supercoiling by gyrase is the following :<br /> # Gyrase binds DNA (gyrase binds more strongly to relaxed or linear DNA than to supercoiled one by a factor of about ten)<br /> # Cleavage of the G-segment<br /> # Passage of T-segment of the other strand through the cleavage site (thanks to ATP)<br /> # Reunion of the G segment<br /> # Translocation<br /> <br /> The '''A subunit is responsible for the breakage reunion of DNA whereas the B-subunit has the ATPase activity.'''<br /> '''The introduction of negative supercoils requires the energy of ATP-hydrolysis. 2 ATP are hydrolyzed per reaction (2 B-subunits in the gyrase)''' so that the linking number (Lk) changes in step of 2. The '''ATPase activity of the B subunit is partially located in the N-terminal region of protein B whereas the C-terminal is involved in the interaction with the A subunit and DNA.''' <br /> '''The B subunit has a weak ATPase activity in the absence of A and DNA, eventhought the A subunit is able to bind DNA without the B subunit. Both subunits are required for all the reactions of gyrase.'''<br /> The DNA supercoiling reaction '''requires in addition to ATP, a divalent cation such as Mg<sup>2+</sup>''', and is stimulated in the presence of spermidine. <br /> <br /> Therefore, the gyrase could also catalyse the following reaction :<br /> * Relaxation of negative/positive supercoils<br /> * Catenation/Decatenation of closed-circular DNA<br /> * DNA cleavage (induced by Quinolone)<br /> * Unknotting DNA strands<br /> <br /> Contrary to the introduction of negative supercoils, '''some of these reactions don't necessarily need the energy from ATP-hydrolysis.'''<br /> <br /> The cleavage of DNA is achieved by a '''transesterification reaction between the tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> and the target phosphoryl groups on opposing strands of the DNA backbone''', resulting in the tyrosine being covalentlty attached to the 5' end of the cleaved segment with a 4-base overhang.<br /> The '''active site tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene>''' are on loops at either end of the dimer interface, 30Å apart, and sit at the ends of strongly basic grooves created by the dimer-related monomers.<br /> <br /> The gyrase structure reveals a '''new cluster of conserved residues''', juxtaposing Tyr 122 and Arg 121 from one monomer and His 80, Arg 32 and Lys 42 from the other monomer. '''This cluster may form the active site of the breakage–reunion reaction''', with the other conserved positive charges (Arg 46 and Arg 47) anchoring the non-covalently bound 3' end of the cleaved DNA.<br /> <br /> The gyrase could be inhibited by both the [http://en.wikipedia.org/wiki/Quinolone quinolones] and [http://en.wikipedia.org/wiki/Coumarin coumarins].<br /> <br /> '''Coumarins prevent the B-subunit of the gyrase from hydrolyzing the ATP''', so that the gyrase can't come back to its starting state for another round of supercoiling. The mecanism is not yet well known.<br /> <br /> '''Quinolones''' inhibit DNA supercoiling by gyrase, it has even the ability to '''induce DNA cleavage''' instead. It appears that '''these drugs will arrest any gyrase rection involving double-strand DNA breakage.'''<br /> However, some mutations offers quinolone resistance, most of them involved a part of the GyrA sequence : between the amino acids 67 and 106. <br /> There are also others antibiotics, like the Cinodine which binds DNA and inhibits its supercoiling by gyrase.<br /> <br /> ===See Also===<br /> *[[Gyrase|Gyrase]]<br /> *[[Topoisomerase|Topoisomerase]]<br /> <br /> ===References===<br /> <ref group="xtra">PMID:009278055</ref><references group="xtra"/><br /> [[Category: Escherichia coli]]<br /> [[Category: Cabral, J H.M.]]<br /> [[Category: Liddington, R C.]]<br /> [[Category: Maxwell, A.]]<br /> [[Category: Gyrase]]<br /> [[Category: Supercoiling dna]]<br /> [[Category: Topoisomerase]]<br /> [[Category: Topoisomerase ii]]<br /> *Reece, Richard J., Anthony Maxwell, and James C. Wang. "DNA gyrase: structure and function." Critical reviews in biochemistry and molecular biology 26.3-4 (1991): 335-375.</div>

Laurent Thomas https://proteopedia.org/index.php?title=Sandbox_Reserved_828&diff=1881690 Sandbox Reserved 828 2014-01-01T11:39:30Z

<p>Laurent Thomas: </p> <hr /> <div><br /> {{Sandbox_Reserved_ESBS}}<br /> <br /> <br /> ----<br /> {{STRUCTURE_1ab4| PDB=1ab4 | SCENE=56/568026/Coloured/1}} <br /> [[Image:1ab4.png|left|200px]]<br /> <br /> =='''Introduction :'''==<br /> <br /> <br /> Gyrase is the '''only prokaryote DNA topoisomerase II able to introduce negative supercoils in the DNA''' (see [http://en.wikipedia.org/wiki/DNA_supercoil DNA Supercoil]) in order to remove positive supercoils. It '''catalyses the hydrolysis of two phosphodiester bonds in a DNA segment (DNA cleavage)''' called G segment. Then, thanks to '''ATP dependant conformation changes''', it enables the passage of another segment (the T segment) through the break, and then religates the broken segment. Gyrase acts prior to the replication (before the replication fork) or other mecanisms requiring loose DNA. <br /> '''In abscence of ATP, like other topoisomerases II, gyrase only relaxes supercoils.'''<br /> <br /> Gyrase is '''coded by two differents contiguous genes gyrA and gyrB''' as it is a 350 kDa '''A<sub>2</sub>B<sub>2</sub> heterotetramers''' of two A proteins and two B proteins. <br /> <br /> The A protein breaks and religates DNA. <br /> The B protein has ATPase activity.<br /> <br /> ----<br /> =='''Structure'''==<br /> <br /> [[Image:Gyrasegene.jpg]]<br /> <br /> '''The A protein''' breaks and religates DNA as the DNA cleavage core and the CTD lies on it. The smallest fragment capable of catalyzing DNA breakage and reunion is between residues 7 and 523 of A sequence, it is the gyrA59 (59kDa). <br /> The gyrA N-ter domain is called '''breakage and reunion domain'''. It is composed of two domains at the <scene name='56/568026/Head/2'>head</scene> region :<br /> <br /> *a winged-helix-turn-helix domain or <scene name='56/568026/Whd/1'>winged helix domain (WHD</scene>) where lies the catalytic tyrosines: the active-site tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> are on loops at either end of the dimer interface.<br /> <br /> *a <scene name='56/568026/Tower/1'>tower domain</scene> with alpha/beta structure that participates in DNA bending during cleavage.<br /> <br /> The '''‘head’ dimer interface is dominated by an antiparallel side by-side packing of the <scene name='56/568026/Alpha3/2'>alpha 3</scene> (the first helix of the HTH motif) from each monomer''', together with their adjoining loops. At the top of the interface, '''the ‘recognition’ helices <scene name='56/568026/Alpha4/1'>alpha 4</scene> make a head-to-head antiparallel dimer contact.'''<br /> <br /> <br /> There is a '''<scene name='56/568026/Coiledcoil_141819/1'>Coiled-coil</scene> domain''', folowing the tower, with a helical core at the tail region. Two long helices (<br /> <scene name='56/568026/Helix14/1'>alpha helix 14</scene> and <scene name='56/568026/Helix18/1'>alpha helix 18</scene>) emanate from this core and connect, together with the C-terminal helix (<br /> <scene name='56/568026/Helix19/1'>alpha helix 19</scene>), the head and tail fragments. This domain has a small globular domain at it's end and is '''involved in the dimerization creating the <scene name='56/568026/Coiledcoil/1'>C-gate</scene>.'''<br /> <br /> <br /> The '''three connecting helices (<scene name='56/568026/Helix14/1'>α14</scene>, <scene name='56/568026/Helix18/1'>α18</scene> and <scene name='56/568026/Helix19/1'>α19</scene>)''' adopt very different conformations, leading to '''large quaternary movements''' involving a single hinge-point within the helices and '''rigid body movements of the head fragments.''' <br /> <br /> [[Image:helixrot.jpg]] [[Image:gyrA592.jpg]]<br /> <br /> The tail is structurally conserved although large surface loops emanating from different points give it a different outward appearance. <br /> '''GyrA forms a heart-shaped homodimer with two protein interfaces, the DNA- and C-gates'''. GyrA59 is the minimal fragment of the A-subunit which, when complexed with the B-subunit, has DNA-cleavage activity. <br /> The remaining 30–35 kDa comprising the C-terminal domain (CTD) of GyrA shows a domain forming a b-pinwheel with a positively charged amino-acid perimeter. This carboxy-terminal domain of GyrA (cyan) is required for the introduction of DNA supercoils), DNA wraps around it. It is linked to the central part of Gyrase A by a flexible 13-15 residues region and stands close to the C-gate and may present an up and down movement in the early stades of the catalytic cycle and may be involved in the helping of G segment and D-gate binding so as the CTD binding of DNA.<br /> <br /> '''The B protein''' has the GHKL ATPase domain, a transducer domain and the Toprim fold on it. <br /> Two ATPase domains from two monomers dimerize to form a closed conformation, the N-gate. <br /> The Toprim fold is a Rossmann fold (beta-alpha-beta-alpha-beta) that contains three invariant acidic residues that coordinate magnesium ions involved in DNA cleavage and DNA religation. This torpim fold interacts with the Winged helix domain of gyrA to create the D-gate.<br /> <br /> <br /> '''The central core''' of the protein is made out of the association of GyrA and Gyr B with the Toprim fold, the DNA-binding domain (WHD)and tower domain, all three creating the D-gate.<br /> These domains are composed of numerous positively charged residues to interact with DNA.<br /> The Toprim domain is flexible and that this flexibility can allow the Toprim domain to coordinate with <br /> the WHD to form a competent cleavage complex.<br /> DNA is bent by ~150 degrees through an invariant isoleucine (I172)<br /> <br /> <br /> [[Image:Gyrasegates.jpg]]<br /> <br /> <br /> ----<br /> =='''Mechanism'''==<br /> <br /> 40-bp of duplex DNA, the G-segment, bind to the core of the enzyme and are cleaved by the active site tyrosines, while another DNA duplex, the T-segment, is captured through the ATP-induced dimerization of a protein gate, the N-gate. After passage through the transiently broken G-segment (DNA gate), the T-segment exits the protein through another protein gate, the C-gate. ATP hydrolysis and release reset the conformation of the enzyme and DNA to their initial state, poised for another strand-passage event or release of the DNA.<br /> N- and C-gates that open or close to allow T-segment transport through both the protein and the cleaved G-segment.<br /> <br /> <br /> [[Image:mecanismgyrase.jpg]]<br /> <br /> ----<br /> <br /> =='''Reaction'''==<br /> <br /> The process of DNA supercoiling by gyrase is the following :<br /> # Gyrase binds DNA (gyrase binds more strongly to relaxed or linear DNA than to supercoiled one by a factor of about ten)<br /> # Cleavage of the G-segment<br /> # Passage of T-segment of the other strand through the cleavage site (thanks to ATP)<br /> # Reunion of the G segment<br /> # Translocation<br /> <br /> The '''A subunit is responsible for the breakage reunion of DNA whereas the B-subunit has the ATPase activity.'''<br /> '''The introduction of negative supercoils requires the energy of ATP-hydrolysis. 2 ATP are hydrolyzed per reaction (2 B-subunits in the gyrase)''' so that the linking number (Lk) changes in step of 2. The '''ATPase activity of the B subunit is partially located in the N-terminal region of protein B whereas the C-terminal is involved in the interaction with the A subunit and DNA.''' <br /> '''The B subunit has a weak ATPase activity in the absence of A and DNA, eventhought the A subunit is able to bind DNA without the B subunit. Both subunits are required for all the reactions of gyrase.'''<br /> The DNA supercoiling reaction '''requires in addition to ATP, a divalent cation such as Mg<sup>2+</sup>''', and is stimulated in the presence of spermidine. <br /> <br /> Therefore, the gyrase could also catalyse the following reaction :<br /> * Relaxation of negative/positive supercoils<br /> * Catenation/Decatenation of closed-circular DNA<br /> * DNA cleavage (induced by Quinolone)<br /> * Unknotting DNA strands<br /> <br /> Contrary to the introduction of negative supercoils, '''some of these reactions don't necessarily need the energy from ATP-hydrolysis.'''<br /> <br /> The cleavage of DNA is achieved by a '''transesterification reaction between the tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> and the target phosphoryl groups on opposing strands of the DNA backbone''', resulting in the tyrosine being covalentlty attached to the 5' end of the cleaved segment with a 4-base overhang.<br /> The '''active site tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene>''' are on loops at either end of the dimer interface, 30Å apart, and sit at the ends of strongly basic grooves created by the dimer-related monomers.<br /> <br /> The gyrase structure reveals a '''new cluster of conserved residues''', juxtaposing Tyr 122 and Arg 121 from one monomer and His 80, Arg 32 and Lys 42 from the other monomer. '''This cluster may form the active site of the breakage–reunion reaction''', with the other conserved positive charges (Arg 46 and Arg 47) anchoring the non-covalently bound 3' end of the cleaved DNA.<br /> <br /> The gyrase could be inhibited by both the [http://en.wikipedia.org/wiki/Quinolone quinolones] and [http://en.wikipedia.org/wiki/Coumarin coumarins].<br /> <br /> '''Coumarins prevent the B-subunit of the gyrase from hydrolyzing the ATP''', so that the gyrase can't come back to its starting state for another round of supercoiling. The mecanism is not yet well known.<br /> <br /> '''Quinolones''' inhibit DNA supercoiling by gyrase, it has even the ability to '''induce DNA cleavage''' instead. It appears that '''these drugs will arrest any gyrase rection involving double-strand DNA breakage.'''<br /> However, some mutations offers quinolone resistance, most of them involved a part of the GyrA sequence : between the amino acids 67 and 106. <br /> There are also others antibiotics, like the Cinodine which binds DNA and inhibits its supercoiling by gyrase.<br /> <br /> ==See Also==<br /> *[[Gyrase|Gyrase]]<br /> *[[Topoisomerase|Topoisomerase]]<br /> <br /> ==Reference==<br /> <ref group="xtra">PMID:009278055</ref><references group="xtra"/><br /> [[Category: Escherichia coli]]<br /> [[Category: Cabral, J H.M.]]<br /> [[Category: Liddington, R C.]]<br /> [[Category: Maxwell, A.]]<br /> [[Category: Gyrase]]<br /> [[Category: Supercoiling dna]]<br /> [[Category: Topoisomerase]]<br /> [[Category: Topoisomerase ii]]<br /> *Reece, Richard J., Anthony Maxwell, and James C. Wang. "DNA gyrase: structure and function." Critical reviews in biochemistry and molecular biology 26.3-4 (1991): 335-375.</div>

Laurent Thomas https://proteopedia.org/index.php?title=Sandbox_Reserved_828&diff=1881689 Sandbox Reserved 828 2014-01-01T11:38:22Z

<p>Laurent Thomas: </p> <hr /> <div><br /> {{Sandbox_Reserved_ESBS}}<br /> <br /> <br /> ----<br /> {{STRUCTURE_1ab4| PDB=1ab4 | SCENE=56/568026/Coloured/1}} <br /> [[Image:1ab4.png|left|200px]]<br /> <br /> =='''Introduction :'''==<br /> <br /> <br /> Gyrase is the '''only prokaryote DNA topoisomerase II able to introduce negative supercoils in the DNA''' (see [http://en.wikipedia.org/wiki/DNA_supercoil DNA Supercoil]) in order to remove positive supercoils. It '''catalyses the hydrolysis of two phosphodiester bonds in a DNA segment (DNA cleavage)''' called G segment. Then, thanks to '''ATP dependant conformation changes''', it enables the passage of another segment (the T segment) through the break, and then religates the broken segment. Gyrase acts prior to the replication (before the replication fork) or other mecanisms requiring loose DNA. <br /> '''In abscence of ATP, like other topoisomerases II, gyrase only relaxes supercoils.'''<br /> <br /> Gyrase is '''coded by two differents contiguous genes gyrA and gyrB''' as it is a 350 kDa '''A<sub>2</sub>B<sub>2</sub> heterotetramers''' of two A proteins and two B proteins. <br /> <br /> The A protein breaks and religates DNA. <br /> The B protein has ATPase activity.<br /> <br /> ----<br /> =='''Structure'''==<br /> <br /> [[Image:Gyrasegene.jpg]]<br /> <br /> '''The A protein''' breaks and religates DNA as the DNA cleavage core and the CTD lies on it. The smallest fragment capable of catalyzing DNA breakage and reunion is between residues 7 and 523 of A sequence, it is the gyrA59 (59kDa). <br /> The gyrA N-ter domain is called '''breakage and reunion domain'''. It is composed of two domains at the <scene name='56/568026/Head/2'>head</scene> region :<br /> <br /> *a winged-helix-turn-helix domain or <scene name='56/568026/Whd/1'>winged helix domain (WHD</scene>) where lies the catalytic tyrosines: the active-site tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> are on loops at either end of the dimer interface.<br /> <br /> *a <scene name='56/568026/Tower/1'>tower domain</scene> with alpha/beta structure that participates in DNA bending during cleavage.<br /> <br /> The '''‘head’ dimer interface is dominated by an antiparallel side by-side packing of the <scene name='56/568026/Alpha3/2'>alpha 3</scene> (the first helix of the HTH motif) from each monomer''', together with their adjoining loops. At the top of the interface, '''the ‘recognition’ helices <scene name='56/568026/Alpha4/1'>alpha 4</scene> make a head-to-head antiparallel dimer contact.'''<br /> <br /> <br /> There is a '''<scene name='56/568026/Coiledcoil_141819/1'>Coiled-coil</scene> domain''', folowing the tower, with a helical core at the tail region. Two long helices (<br /> <scene name='56/568026/Helix14/1'>alpha helix 14</scene> and <scene name='56/568026/Helix18/1'>alpha helix 18</scene>) emanate from this core and connect, together with the C-terminal helix (<br /> <scene name='56/568026/Helix19/1'>alpha helix 19</scene>), the head and tail fragments. This domain has a small globular domain at it's end and is '''involved in the dimerization creating the <scene name='56/568026/Coiledcoil/1'>C-gate</scene>.'''<br /> <br /> <br /> The '''three connecting helices (<scene name='56/568026/Helix14/1'>α14</scene>, <scene name='56/568026/Helix18/1'>α18</scene> and <scene name='56/568026/Helix19/1'>α19</scene>)''' adopt very different conformations, leading to '''large quaternary movements''' involving a single hinge-point within the helices and '''rigid body movements of the head fragments.''' <br /> <br /> [[Image:helixrot.jpg]] [[Image:gyrA592.jpg]]<br /> <br /> The tail is structurally conserved although large surface loops emanating from different points give it a different outward appearance. <br /> '''GyrA forms a heart-shaped homodimer with two protein interfaces, the DNA- and C-gates'''. GyrA59 is the minimal fragment of the A-subunit which, when complexed with the B-subunit, has DNA-cleavage activity. <br /> The remaining 30–35 kDa comprising the C-terminal domain (CTD) of GyrA shows a domain forming a b-pinwheel with a positively charged amino-acid perimeter. This carboxy-terminal domain of GyrA (cyan) is required for the introduction of DNA supercoils), DNA wraps around it. It is linked to the central part of Gyrase A by a flexible 13-15 residues region and stands close to the C-gate and may present an up and down movement in the early stades of the catalytic cycle and may be involved in the helping of G segment and D-gate binding so as the CTD binding of DNA.<br /> <br /> '''The B protein''' has the GHKL ATPase domain, a transducer domain and the Toprim fold on it. <br /> Two ATPase domains from two monomers dimerize to form a closed conformation, the N-gate. <br /> The Toprim fold is a Rossmann fold (beta-alpha-beta-alpha-beta) that contains three invariant acidic residues that coordinate magnesium ions involved in DNA cleavage and DNA religation. This torpim fold interacts with the Winged helix domain of gyrA to create the D-gate.<br /> <br /> <br /> '''The central core''' of the protein is made out of the association of GyrA and Gyr B with the Toprim fold, the DNA-binding domain (WHD)and tower domain, all three creating the D-gate.<br /> These domains are composed of numerous positively charged residues to interact with DNA.<br /> The Toprim domain is flexible and that this flexibility can allow the Toprim domain to coordinate with <br /> the WHD to form a competent cleavage complex.<br /> DNA is bent by ~150 degrees through an invariant isoleucine (I172)<br /> <br /> <br /> [[Image:Gyrasegates.jpg]]<br /> <br /> <br /> ----<br /> =='''Mechanism'''==<br /> <br /> 40-bp of duplex DNA, the G-segment, bind to the core of the enzyme and are cleaved by the active site tyrosines, while another DNA duplex, the T-segment, is captured through the ATP-induced dimerization of a protein gate, the N-gate. After passage through the transiently broken G-segment (DNA gate), the T-segment exits the protein through another protein gate, the C-gate. ATP hydrolysis and release reset the conformation of the enzyme and DNA to their initial state, poised for another strand-passage event or release of the DNA.<br /> N- and C-gates that open or close to allow T-segment transport through both the protein and the cleaved G-segment.<br /> <br /> <br /> [[Image:mecanismgyrase.jpg]]<br /> <br /> ----<br /> <br /> =='''Reaction'''==<br /> <br /> The process of DNA supercoiling by gyrase is the following :<br /> # Gyrase binds DNA (gyrase binds more strongly to relaxed or linear DNA than to supercoiled one by a factor of about ten)<br /> # Cleavage of the G-segment<br /> # Passage of T-segment of the other strand through the cleavage site (thanks to ATP)<br /> # Reunion of the G segment<br /> # Translocation<br /> <br /> The '''A subunit is responsible for the breakage reunion of DNA whereas the B-subunit has the ATPase activity.'''<br /> '''The introduction of negative supercoils requires the energy of ATP-hydrolysis. 2 ATP are hydrolyzed per reaction (2 B-subunits in the gyrase)''' so that the linking number (Lk) changes in step of 2. The '''ATPase activity of the B subunit is partially located in the N-terminal region of protein B whereas the C-terminal is involved in the interaction with the A subunit and DNA.''' <br /> '''The B subunit has a weak ATPase activity in the absence of A and DNA, eventhought the A subunit is able to bind DNA without the B subunit. Both subunits are required for all the reactions of gyrase.'''<br /> The DNA supercoiling reaction '''requires in addition to ATP, a divalent cation such as Mg<sup>2+</sup>''', and is stimulated in the presence of spermidine. <br /> <br /> Therefore, the gyrase could also catalyse the following reaction :<br /> * Relaxation of negative/positive supercoils<br /> * Catenation/Decatenation of closed-circular DNA<br /> * DNA cleavage (induced by Quinolone)<br /> * Unknotting DNA strands<br /> <br /> Contrary to the introduction of negative supercoils, some of these reactions don't necessarily need the energy from ATP-hydrolysis.<br /> <br /> The cleavage of DNA is achieved by a '''transesterification reaction between the tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> and the target phosphoryl groups on opposing strands of the DNA backbone''', resulting in the tyrosine being covalentlty attached to the 5' end of the cleaved segment with a 4-base overhang.<br /> The '''active site tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene>''' are on loops at either end of the dimer interface, 30Å apart, and sit at the ends of strongly basic grooves created by the dimer-related monomers.<br /> <br /> The gyrase structure reveals a '''new cluster of conserved residues''', juxtaposing Tyr 122 and Arg 121 from one monomer and His 80, Arg 32 and Lys 42 from the other monomer. '''This cluster may form the active site of the breakage–reunion reaction''', with the other conserved positive charges (Arg 46 and Arg 47) anchoring the non-covalently bound 3' end of the cleaved DNA.<br /> <br /> The gyrase could be inhibited by both the [http://en.wikipedia.org/wiki/Quinolone quinolones] and [http://en.wikipedia.org/wiki/Coumarin coumarins].<br /> <br /> '''Coumarins prevent the B-subunit of the gyrase from hydrolyzing the ATP''', so that the gyrase can't come back to its starting state for another round of supercoiling. The mecanism is not yet well known.<br /> <br /> '''Quinolones''' inhibit DNA supercoiling by gyrase, it has even the ability to '''induce DNA cleavage''' instead. It appears that '''these drugs will arrest any gyrase rection involving double-strand DNA breakage.'''<br /> However, some mutations offers quinolone resistance, most of them involved a part of the GyrA sequence : between the amino acids 67 and 106. <br /> There are also others antibiotics, like the Cinodine which binds DNA and inhibits its supercoiling by gyrase.<br /> <br /> ==See Also==<br /> *[[Gyrase|Gyrase]]<br /> *[[Topoisomerase|Topoisomerase]]<br /> <br /> ==Reference==<br /> <ref group="xtra">PMID:009278055</ref><references group="xtra"/><br /> [[Category: Escherichia coli]]<br /> [[Category: Cabral, J H.M.]]<br /> [[Category: Liddington, R C.]]<br /> [[Category: Maxwell, A.]]<br /> [[Category: Gyrase]]<br /> [[Category: Supercoiling dna]]<br /> [[Category: Topoisomerase]]<br /> [[Category: Topoisomerase ii]]<br /> *Reece, Richard J., Anthony Maxwell, and James C. Wang. "DNA gyrase: structure and function." Critical reviews in biochemistry and molecular biology 26.3-4 (1991): 335-375.</div>

Laurent Thomas https://proteopedia.org/index.php?title=Sandbox_Reserved_828&diff=1881381 Sandbox Reserved 828 2013-12-30T22:01:49Z

<p>Laurent Thomas: </p> <hr /> <div><br /> {{Sandbox_Reserved_ESBS}}<br /> <br /> <br /> ----<br /> {{STRUCTURE_1ab4| PDB=1ab4 | SCENE=56/568026/Coloured/1}} <br /> [[Image:1ab4.png|left|200px]]<br /> <br /> =='''Introduction :'''==<br /> <br /> <br /> Gyrase is the '''only prokaryote DNA topoisomerase II able to introduce negative supercoils in the DNA''' (see [http://en.wikipedia.org/wiki/DNA_supercoil DNA Supercoil]) in order to remove positive supercoils. It '''catalyses the hydrolysis of two phosphodiester bonds in a DNA segment (DNA cleavage)''' called G segment. Then, thanks to '''ATP dependant conformation changes''', it enables the passage of another segment (the T segment) through the break, and then religates the broken segment. Gyrase acts prior to the replication (before the replication fork) or other mecanisms requiring loose DNA. <br /> '''In abscence of ATP, like other topoisomerases II, gyrase only relaxes supercoils.'''<br /> <br /> Gyrase is '''coded by two differents contiguous genes gyrA and gyrB''' as it is a 350 kDa '''A<sub>2</sub>B<sub>2</sub> heterotetramers''' of two A proteins and two B proteins. <br /> <br /> The A protein breaks and religates DNA. <br /> The B protein has ATPase activity.<br /> <br /> ----<br /> =='''Structure'''==<br /> <br /> [[Image:Gyrasegene.jpg]]<br /> <br /> '''The A protein''' breaks and religates DNA as the DNA cleavage core and the CTD lies on it. The smallest fragment capable of catalyzing DNA breakage and reunion is between residues 7 and 523 of A sequence, it is the gyrA59 (59kDa). <br /> The gyrA N-ter domain is called '''breakage and reunion domain'''. It is composed of two domains at the <scene name='56/568026/Head/2'>head</scene> region :<br /> <br /> *a winged-helix-turn-helix domain or <scene name='56/568026/Whd/1'>winged helix domain (WHD</scene>) where lies the catalytic tyrosines: the active-site tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> are on loops at either end of the dimer interface.<br /> <br /> *a <scene name='56/568026/Tower/1'>tower domain</scene> with alpha/beta structure that participates in DNA bending during cleavage.<br /> <br /> The '''‘head’ dimer interface is dominated by an antiparallel side by-side packing of the <scene name='56/568026/Alpha3/2'>alpha 3</scene> (the first helix of the HTH motif) from each monomer''', together with their adjoining loops. At the top of the interface, '''the ‘recognition’ helices <scene name='56/568026/Alpha4/1'>alpha 4</scene> make a head-to-head antiparallel dimer contact.'''<br /> <br /> <br /> There is a '''<scene name='56/568026/Coiledcoil_141819/1'>Coiled-coil</scene> domain''', folowing the tower, with a helical core at the tail region. Two long helices (<br /> <scene name='56/568026/Helix14/1'>alpha helix 14</scene> and <scene name='56/568026/Helix18/1'>alpha helix 18</scene>) emanate from this core and connect, together with the C-terminal helix (<br /> <scene name='56/568026/Helix19/1'>alpha helix 19</scene>), the head and tail fragments. This domain has a small globular domain at it's end and is '''involved in the dimerization creating the <scene name='56/568026/Coiledcoil/1'>C-gate</scene>.'''<br /> <br /> <br /> The '''three connecting helices (<scene name='56/568026/Helix14/1'>α14</scene>, <scene name='56/568026/Helix18/1'>α18</scene> and <scene name='56/568026/Helix19/1'>α19</scene>)''' adopt very different conformations, leading to '''large quaternary movements''' involving a single hinge-point within the helices and '''rigid body movements of the head fragments.''' <br /> <br /> [[Image:helixrot.jpg]] [[Image:gyrA592.jpg]]<br /> <br /> The tail is structurally conserved although large surface loops emanating from different points give it a different outward appearance. <br /> '''GyrA forms a heart-shaped homodimer with two protein interfaces, the DNA- and C-gates'''. GyrA59 is the minimal fragment of the A-subunit which, when complexed with the B-subunit, has DNA-cleavage activity. <br /> The remaining 30–35 kDa comprising the C-terminal domain (CTD) of GyrA shows a domain forming a b-pinwheel with a positively charged amino-acid perimeter. This carboxy-terminal domain of GyrA (cyan) is required for the introduction of DNA supercoils), DNA wraps around it. It is linked to the central part of Gyrase A by a flexible 13-15 residues region and stands close to the C-gate and may present an up and down movement in the early stades of the catalytic cycle and may be involved in the helping of G segment and D-gate binding so as the CTD binding of DNA.<br /> <br /> '''The B protein''' has the GHKL ATPase domain, a transducer domain and the Toprim fold on it. <br /> Two ATPase domains from two monomers dimerize to form a closed conformation, the N-gate. <br /> The Toprim fold is a Rossmann fold (beta-alpha-beta-alpha-beta) that contains three invariant acidic residues that coordinate magnesium ions involved in DNA cleavage and DNA religation. This torpim fold interacts with the Winged helix domain of gyrA to create the D-gate.<br /> <br /> <br /> '''The central core''' of the protein is made out of the association of GyrA and Gyr B with the Toprim fold, the DNA-binding domain (WHD)and tower domain, all three creating the D-gate.<br /> These domains are composed of numerous positively charged residues to interact with DNA.<br /> The Toprim domain is flexible and that this flexibility can allow the Toprim domain to coordinate with <br /> the WHD to form a competent cleavage complex.<br /> DNA is bent by ~150 degrees through an invariant isoleucine (I172)<br /> <br /> <br /> [[Image:Gyrasegates.jpg]]<br /> <br /> <br /> ----<br /> =='''Mechanism'''==<br /> <br /> 40-bp of duplex DNA, the G-segment, bind to the core of the enzyme and are cleaved by the active site tyrosines, while another DNA duplex, the T-segment, is captured through the ATP-induced dimerization of a protein gate, the N-gate. After passage through the transiently broken G-segment (DNA gate), the T-segment exits the protein through another protein gate, the C-gate. ATP hydrolysis and release reset the conformation of the enzyme and DNA to their initial state, poised for another strand-passage event or release of the DNA.<br /> N- and C-gates that open or close to allow T-segment transport through both the protein and the cleaved G-segment.<br /> <br /> <br /> [[Image:mecanismgyrase.jpg]]<br /> <br /> ----<br /> <br /> =='''Reaction'''==<br /> <br /> The process of DNA supercoiling by gyrase is the following :<br /> # Gyrase binds DNA (gyrase binds more strongly to relaxed or linear DNA than to supercoiled one by a factor of about ten)<br /> # Cleavage of the G-segment<br /> # Passage of T-segment of the other strand through the cleavage site (thanks to ATP)<br /> # Reunion of the G segment<br /> # Translocation<br /> <br /> The A subunit is responsible for the breakage reunion of DNA whereas the B-subunit has the ATPase activity.<br /> The introduction of negative supercoils requires the energy of ATP-hydrolysis. 2 ATP are hydrolyzed per reaction (2 B-subunits in the gyrase) so that the linking number (Lk) changes in step of 2. The ATPase activity of the B subunit is partially located in the N-terminal region of protein B whereas the C-terminal is involved in the interaction with the A subunit and DNA. <br /> The B subunit has a weak ATPase activity in the absence of A and DNA, eventhought the A subunit is able to bind DNA without the B subunit. Both subunits are required for all the reactions of gyrase.<br /> The DNA supercoiling reaction requires in addition to ATP, a divalent cation such as Mg<sup>2+</sup>, and is stimulated in the presence of spermidine. <br /> Therefore, the gyrase could also catalyse the following reaction :<br /> * Relaxation of negative/positive supercoils<br /> * Catenation/Decatenation of closed-circular DNA<br /> * DNA cleavage (induced by Quinolone)<br /> * Unknotting DNA strands<br /> <br /> Contrary to the introduction of negative supercoils, some of these reactions don't necessarily need the energy from ATP-hydrolysis.<br /> <br /> The cleavage of DNA is achieved by a '''transesterification reaction between the tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> and the target phosphoryl groups on opposing strands of the DNA backbone''', resulting in the tyrosine being covalentlty attached to the 5' end of the cleaved segment with a 4-base overhang.<br /> The '''active site tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene>''' are on loops at either end of the dimer interface, 30Å apart, and sit at the ends of strongly basic grooves created by the dimer-related monomers.<br /> <br /> The gyrase structure reveals a '''new cluster of conserved residues''', juxtaposing Tyr 122 and Arg 121 from one monomer and His 80, Arg 32 and Lys 42 from the other monomer. '''This cluster may form the active site of the breakage–reunion reaction''', with the other conserved positive charges (Arg 46 and Arg 47) anchoring the non-covalently bound 3' end of the cleaved DNA.<br /> <br /> The gyrase could be inhibited by both the quinolones and the coumarins.<br /> Coumarins prevent the B-subunit of the gyrase from hydrolyzing the ATP, so that the gyrase can't come back to its starting state for another round of supercoiling. The mecanism is not yet well known.<br /> The quinolones inhibit DNA supercoiling by gyrase, it has even the ability to induce DNA cleavage instead. It appears that these drugs will arrest any gyrase rection involving double-strand DNA breakage.<br /> However, some mutations offers quinolone resistance, most of them involved a part of the GyrA sequence : between the amino acid 67 and 106. <br /> There are also others antibiotics, like the Cinodine which binds DNA and inhibits its supercoiling by gyrase.<br /> <br /> ==See Also==<br /> *[[Gyrase|Gyrase]]<br /> *[[Topoisomerase|Topoisomerase]]<br /> <br /> ==Reference==<br /> <ref group="xtra">PMID:009278055</ref><references group="xtra"/><br /> [[Category: Escherichia coli]]<br /> [[Category: Cabral, J H.M.]]<br /> [[Category: Liddington, R C.]]<br /> [[Category: Maxwell, A.]]<br /> [[Category: Gyrase]]<br /> [[Category: Supercoiling dna]]<br /> [[Category: Topoisomerase]]<br /> [[Category: Topoisomerase ii]]<br /> *Reece, Richard J., Anthony Maxwell, and James C. Wang. "DNA gyrase: structure and function." Critical reviews in biochemistry and molecular biology 26.3-4 (1991): 335-375.</div>

Laurent Thomas https://proteopedia.org/index.php?title=Sandbox_Reserved_828&diff=1881380 Sandbox Reserved 828 2013-12-30T21:54:43Z

<p>Laurent Thomas: </p> <hr /> <div><br /> {{Sandbox_Reserved_ESBS}}<br /> <br /> <br /> ----<br /> {{STRUCTURE_1ab4| PDB=1ab4 | SCENE=56/568026/Coloured/1}} <br /> [[Image:1ab4.png|left|200px]]<br /> <br /> =='''Introduction :'''==<br /> <br /> <br /> Gyrase is the '''only prokaryote DNA topoisomerase II able to introduce negative supercoils in the DNA''' (see [http://en.wikipedia.org/wiki/DNA_supercoil DNA Supercoil]) in order to remove positive supercoils. It '''catalyses the hydrolysis of two phosphodiester bonds in a DNA segment (DNA cleavage)''' called G segment. Then, thanks to '''ATP dependant conformation changes''', it enables the passage of another segment (the T segment) through the break, and then religates the broken segment. Gyrase acts prior to the replication (before the replication fork) or other mecanisms requiring loose DNA. <br /> '''In abscence of ATP, like other topoisomerases II, gyrase only relaxes supercoils.'''<br /> <br /> Gyrase is '''coded by two differents contiguous genes gyrA and gyrB''' as it is a 350 kDa '''A<sub>2</sub>B<sub>2</sub> heterotetramers''' of two A proteins and two B proteins. <br /> <br /> The A protein breaks and religates DNA. <br /> The B protein has ATPase activity.<br /> <br /> ----<br /> =='''Structure'''==<br /> <br /> [[Image:Gyrasegene.jpg]]<br /> <br /> '''The A protein''' breaks and religates DNA as the DNA cleavage core and the CTD lies on it. The smallest fragment capable of catalyzing DNA breakage and reunion is between residues 7 and 523 of A sequence, it is the gyrA59 (59kDa). <br /> The gyrA N-ter domain is called '''breakage and reunion domain'''. It is composed of two domains at the <scene name='56/568026/Head/2'>head</scene> region :<br /> <br /> *a winged-helix-turn-helix domain or <scene name='56/568026/Whd/1'>winged helix domain (WHD</scene>) where lies the catalytic tyrosines: the active-site tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> are on loops at either end of the dimer interface.<br /> <br /> *a <scene name='56/568026/Tower/1'>tower domain</scene> with alpha/beta structure that participates in DNA bending during cleavage.<br /> <br /> The '''‘head’ dimer interface is dominated by an antiparallel side by-side packing of the <scene name='56/568026/Alpha3/2'>alpha 3</scene> (the first helix of the HTH motif) from each monomer''', together with their adjoining loops. At the top of the interface, '''the ‘recognition’ helices <scene name='56/568026/Alpha4/1'>alpha 4</scene> make a head-to-head antiparallel dimer contact.'''<br /> <br /> <br /> There is a '''<scene name='56/568026/Coiledcoil_141819/1'>Coiled-coil</scene> domain''', folowing the tower, with a helical core at the tail region. Two long helices (<br /> <scene name='56/568026/Helix14/1'>alpha helix 14</scene> and <scene name='56/568026/Helix18/1'>alpha helix 18</scene>) emanate from this core and connect, together with the C-terminal helix (<br /> <scene name='56/568026/Helix19/1'>alpha helix 19</scene>), the head and tail fragments. This domain has a small globular domain at it's end and is '''involved in the dimerization creating the <scene name='56/568026/Coiledcoil/1'>C-gate</scene>.'''<br /> <br /> <br /> The '''three connecting helices (<scene name='56/568026/Helix14/1'>α14</scene>, <scene name='56/568026/Helix18/1'>α18</scene> and <scene name='56/568026/Helix19/1'>α19</scene>)''' adopt very different conformations, leading to '''large quaternary movements''' involving a single hinge-point within the helices and '''rigid body movements of the head fragments.''' <br /> <br /> [[Image:helixrot.jpg]] [[Image:gyrA592.jpg]]<br /> <br /> The tail is structurally conserved although large surface loops emanating from different points give it a different outward appearance. <br /> GyrA forms a heart-shaped homodimer with two protein interfaces, the DNA- and C-gates. GyrA59 is the minimal fragment of the A-subunit which, when complexed with the B-subunit, has DNA-cleavage activity. <br /> The remaining 30–35 kDa comprising the C-terminal domain (CTD) of GyrA shows a domain forming a b-pinwheel with a positively charged amino-acid perimeter. This carboxy-terminal domain of GyrA (cyan) is required for the introduction of DNA supercoils), DNA wraps around it. It is linked to the central part of Gyrase A by a flexible 13-15 residues region and stands close to the C-gate and may present an up and down movement in the early stades of the catalytic cycle and may be involved in the helping of G segment and D-gate binding so as the CTD binding of DNA.<br /> <br /> '''The B protein''' has the GHKL ATPase domain, a transducer domain and the Toprim fold on it. <br /> Two ATPase domains from two monomers dimerize to form a closed conformation, the N-gate. <br /> The Toprim fold is a Rossmann fold (beta-alpha-beta-alpha-beta) that contains three invariant acidic residues that coordinate magnesium ions involved in DNA cleavage and DNA religation. This torpim fold interacts with the Winged helix domain of gyrA to create the D-gate.<br /> <br /> <br /> '''The central core''' of the protein is made out of the association of GyrA and Gyr B with the Toprim fold, the DNA-binding domain (WHD)and tower domain, all three creating the D-gate.<br /> These domains are composed of numerous positively charged residues to interact with DNA.<br /> The Toprim domain is flexible and that this flexibility can allow the Toprim domain to coordinate with <br /> the WHD to form a competent cleavage complex.<br /> DNA is bent by ~150 degrees through an invariant isoleucine (I172)<br /> <br /> <br /> [[Image:Gyrasegates.jpg]]<br /> <br /> <br /> ----<br /> '''Mecanism'''<br /> <br /> 40-bp of duplex DNA, the G-segment, bind to the core of the enzyme and are cleaved by the active site tyrosines, while another DNA duplex, the T-segment, is captured through the ATP-induced dimerization of a protein gate, the N-gate. After passage through the transiently broken G-segment (DNA gate), the T-segment exits the protein through another protein gate, the C-gate. ATP hydrolysis and release reset the conformation of the enzyme and DNA to their initial state, poised for another strand-passage event or release of the DNA.<br /> N- and C-gates that open or close to allow T-segment transport through both theprotein and the cleaved G-segment.<br /> <br /> <br /> [[Image:mecanismgyrase.jpg]]<br /> <br /> ----<br /> <br /> =='''Reaction'''==<br /> <br /> The process of DNA supercoiling by gyrase is the following :<br /> # Gyrase binds DNA (gyrase binds more strongly to relaxed or linear DNA than to supercoiled one by a factor of about ten)<br /> # Cleavage of the G-segment<br /> # Passage of T-segment of the other strand through the cleavage site (thanks to ATP)<br /> # Reunion of the G segment<br /> # Translocation<br /> <br /> The A subunit is responsible for the breakage reunion of DNA whereas the B-subunit has the ATPase activity.<br /> The introduction of negative supercoils requires the energy of ATP-hydrolysis. 2 ATP are hydrolyzed per reaction (2 B-subunits in the gyrase)so that the linking number (Lk) changes in step of 2. The ATPase activity of the B subunit is partially located in the N-terminal region of protein B whereas the C-terminal is involved in the interaction with the A subunit and DNA. <br /> The B subunit has a weak ATPase activity in the absence of A and DNA, eventhought the A subunit is able to bind DNA without the B subunit. Both subunits are required for all the reactions of gyrase.<br /> The DNA supercoiling reaction requires in addition to ATP, a divalent cation such as Mg<sup>2+</sup>, and is stimulated in the presence of spermidine. <br /> Therefore, the gyrase could also catalyse the following reaction :<br /> * Relaxation of negative/positive supercoils<br /> * Catenation/Decatenation of closed-circular DNA<br /> * DNA cleavage (induced by Quinolone)<br /> * Unknotting DNA strands<br /> <br /> Contrary to the introduction of negative supercoils, some of these reactions don't necessarily need the energy from ATP-hydrolysis.<br /> <br /> The cleavage of DNA is achieved by a '''transesterification reaction between the tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> and the target phosphoryl groups on opposing strands of the DNA backbone''', resulting in the tyrosine being covalentlty attached to the 5' end of the cleaved segment with a 4-base overhang.<br /> The '''active site tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene>''' are on loops at either end of the dimer interface, 30Å apart, and sit at the ends of strongly basic grooves created by the dimer-related monomers.<br /> <br /> The gyrase structure reveals a '''new cluster of conserved residues''', juxtaposing Tyr 122 and Arg 121 from one monomer and His 80, Arg 32 and Lys 42 from the other monomer. '''This cluster may form the active site of the breakage–reunion reaction''', with the other conserved positive charges (Arg 46 and Arg 47) anchoring the non-covalently bound 3' end of the cleaved DNA.<br /> <br /> The gyrase could be inhibited by both the quinolones and the coumarins.<br /> Coumarins prevent the B-subunit of the gyrase from hydrolyzing the ATP, so that the gyrase can't come back to its starting state for another round of supercoiling. The mecanism is not yet well known.<br /> The quinolones inhibit DNA supercoiling by gyrase, it has even the ability to induce DNA cleavage instead. It appears that these drugs will arrest any gyrase rection involving double-strand DNA breakage.<br /> However, some mutations offers quinolone resistance, most of them involved a part of the GyrA sequence : between the amino acid 67 and 106. <br /> There are also others antibiotics, like the Cinodine which binds DNA and inhibits its supercoiling by gyrase.<br /> <br /> ==See Also==<br /> *[[Gyrase|Gyrase]]<br /> *[[Topoisomerase|Topoisomerase]]<br /> <br /> ==Reference==<br /> <ref group="xtra">PMID:009278055</ref><references group="xtra"/><br /> [[Category: Escherichia coli]]<br /> [[Category: Cabral, J H.M.]]<br /> [[Category: Liddington, R C.]]<br /> [[Category: Maxwell, A.]]<br /> [[Category: Gyrase]]<br /> [[Category: Supercoiling dna]]<br /> [[Category: Topoisomerase]]<br /> [[Category: Topoisomerase ii]]<br /> *Reece, Richard J., Anthony Maxwell, and James C. Wang. "DNA gyrase: structure and function." Critical reviews in biochemistry and molecular biology 26.3-4 (1991): 335-375.</div>

Laurent Thomas https://proteopedia.org/index.php?title=Sandbox_Reserved_828&diff=1881379 Sandbox Reserved 828 2013-12-30T21:45:46Z

<p>Laurent Thomas: </p> <hr /> <div><br /> {{Sandbox_Reserved_ESBS}}<br /> <br /> <br /> ----<br /> {{STRUCTURE_1ab4| PDB=1ab4 | SCENE=56/568026/Coloured/1}} <br /> [[Image:1ab4.png|left|200px]]<br /> <br /> =='''Introduction :'''==<br /> <br /> <br /> Gyrase is the '''only prokaryote DNA topoisomerase II able to introduce negative supercoils in the DNA''' (see [http://en.wikipedia.org/wiki/DNA_supercoil DNA Supercoil]) in order to remove positive supercoils. It '''catalyses the hydrolysis of two phosphodiester bonds in a DNA segment (DNA cleavage)''' called G segment. Then, thanks to '''ATP dependant conformation changes''', it enables the passage of another segment (the T segment) through the break, and then religates the broken segment. Gyrase acts prior to the replication (before the replication fork) or other mecanisms requiring loose DNA. <br /> '''In abscence of ATP, like other topoisomerases II, gyrase only relaxes supercoils.'''<br /> <br /> Gyrase is '''coded by two differents contiguous genes gyrA and gyrB''' as it is a 350 kDa '''A<sub>2</sub>B<sub>2</sub> heterotetramers''' of two A proteins and two B proteins. <br /> <br /> The A protein breaks and religates DNA. <br /> The B protein has ATPase activity.<br /> <br /> ----<br /> =='''Structure'''==<br /> <br /> [[Image:Gyrasegene.jpg]]<br /> <br /> '''The A protein''' breaks and religates DNA as the DNA cleavage core and the CTD lies on it. The smallest fragment capable of catalyzing DNA breakage and reunion is between residues 7 and 523 of A sequence, it is the gyrA59 (59kDa). <br /> The gyrA N-ter domain is called '''breakage and reunion domain'''. It is composed of two domains at the <scene name='56/568026/Head/2'>head</scene> region :<br /> <br /> *a winged-helix-turn-helix domain or <scene name='56/568026/Whd/1'>winged helix domain (WHD</scene>) where lies the catalytic tyrosines: the active-site tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> are on loops at either end of the dimer interface.<br /> <br /> *a <scene name='56/568026/Tower/1'>tower domain</scene> with alpha/beta structure that participates in DNA bending during cleavage.<br /> <br /> The ‘head’ dimer interface is dominated by an antiparallel side by-side packing of the <scene name='56/568026/Alpha3/2'>alpha 3</scene> (the first helix of the HTH motif) from each monomer, together with their adjoining loops. At the top of the interface, the ‘recognition’ helices <scene name='56/568026/Alpha4/1'>alpha4</scene> make a head-to-head antiparallel dimer contact.<br /> <br /> <br /> There is a <scene name='56/568026/Coiledcoil_141819/1'>Coiled-coil</scene> domain, folowing the tower, with a helical core at the tail region. Two long helices (<br /> <scene name='56/568026/Helix14/1'>alpha helix 14</scene> and <scene name='56/568026/Helix18/1'>alpha helix 18</scene>) emanate from this core and connect, together with the C-terminal helix (<br /> <scene name='56/568026/Helix19/1'>alpha helix 19</scene>), the head and tail fragments. This domain has a small globular domain at it's end and is involved in the dimerization creating the <scene name='56/568026/Coiledcoil/1'>C-gate</scene> .<br /> <br /> <br /> The three connecting helices (α14,α18 and α19) adopt very different conformations, leading to large quaternary movements involving a single hinge-point within the helices and rigid body movements of <br /> the head fragments. <br /> <br /> [[Image:helixrot.jpg]] [[Image:gyrA592.jpg]]<br /> <br /> The tail is structurally conserved although large surface loops emanating from different points give it a different outward appearance. <br /> GyrA forms a heart-shaped homodimer with two protein interfaces, the DNA- and C-gates. GyrA59 is the minimal fragment of the A-subunit which, when complexed with the B-subunit, has DNA-cleavage activity. <br /> The remaining 30–35 kDa comprising the C-terminal domain (CTD) of GyrA shows a domain forming a b-pinwheel with a positively charged amino-acid perimeter. This carboxy-terminal domain of GyrA (cyan) is required for the introduction of DNA supercoils), DNA wraps around it. It is linked to the central part of Gyrase A by a flexible 13-15 residues region and stands close to the C-gate and may present an up and down movement in the early stades of the catalytic cycle and may be involved in the helping of G segment and D-gate binding so as the CTD binding of DNA.<br /> <br /> '''The B protein''' has the GHKL ATPase domain, a transducer domain and the Toprim fold on it. <br /> Two ATPase domains from two monomers dimerize to form a closed conformation, the N-gate. <br /> The Toprim fold is a Rossmann fold (beta-alpha-beta-alpha-beta) that contains three invariant acidic residues that coordinate magnesium ions involved in DNA cleavage and DNA religation. This torpim fold interacts with the Winged helix domain of gyrA to create the D-gate.<br /> <br /> <br /> '''The central core''' of the protein is made out of the association of GyrA and Gyr B with the Toprim fold, the DNA-binding domain (WHD)and tower domain, all three creating the D-gate.<br /> These domains are composed of numerous positively charged residues to interact with DNA.<br /> The Toprim domain is flexible and that this flexibility can allow the Toprim domain to coordinate with <br /> the WHD to form a competent cleavage complex.<br /> DNA is bent by ~150 degrees through an invariant isoleucine (I172)<br /> <br /> <br /> [[Image:Gyrasegates.jpg]]<br /> <br /> <br /> ----<br /> '''Mecanism'''<br /> <br /> 40-bp of duplex DNA, the G-segment, bind to the core of the enzyme and are cleaved by the active site tyrosines, while another DNA duplex, the T-segment, is captured through the ATP-induced dimerization of a protein gate, the N-gate. After passage through the transiently broken G-segment (DNA gate), the T-segment exits the protein through another protein gate, the C-gate. ATP hydrolysis and release reset the conformation of the enzyme and DNA to their initial state, poised for another strand-passage event or release of the DNA.<br /> N- and C-gates that open or close to allow T-segment transport through both theprotein and the cleaved G-segment.<br /> <br /> <br /> [[Image:mecanismgyrase.jpg]]<br /> <br /> ----<br /> <br /> =='''Reaction'''==<br /> <br /> The process of DNA supercoiling by gyrase is the following :<br /> # Gyrase binds DNA (gyrase binds more strongly to relaxed or linear DNA than to supercoiled one by a factor of about ten)<br /> # Cleavage of the G-segment<br /> # Passage of T-segment of the other strand through the cleavage site (thanks to ATP)<br /> # Reunion of the G segment<br /> # Translocation<br /> <br /> The A subunit is responsible for the breakage reunion of DNA whereas the B-subunit has the ATPase activity.<br /> The introduction of negative supercoils requires the energy of ATP-hydrolysis. 2 ATP are hydrolyzed per reaction (2 B-subunits in the gyrase)so that the linking number (Lk) changes in step of 2. The ATPase activity of the B subunit is partially located in the N-terminal region of protein B whereas the C-terminal is involved in the interaction with the A subunit and DNA. <br /> The B subunit has a weak ATPase activity in the absence of A and DNA, eventhought the A subunit is able to bind DNA without the B subunit. Both subunits are required for all the reactions of gyrase.<br /> The DNA supercoiling reaction requires in addition to ATP, a divalent cation such as Mg<sup>2+</sup>, and is stimulated in the presence of spermidine. <br /> Therefore, the gyrase could also catalyse the following reaction :<br /> * Relaxation of negative/positive supercoils<br /> * Catenation/Decatenation of closed-circular DNA<br /> * DNA cleavage (induced by Quinolone)<br /> * Unknotting DNA strands<br /> <br /> Contrary to the introduction of negative supercoils, some of these reactions don't necessarily need the energy from ATP-hydrolysis.<br /> <br /> The cleavage of DNA is achieved by a '''transesterification reaction between the tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> and the target phosphoryl groups on opposing strands of the DNA backbone''', resulting in the tyrosine being covalentlty attached to the 5' end of the cleaved segment with a 4-base overhang.<br /> The '''active site tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene>''' are on loops at either end of the dimer interface, 30Å apart, and sit at the ends of strongly basic grooves created by the dimer-related monomers.<br /> <br /> The gyrase structure reveals a '''new cluster of conserved residues''', juxtaposing Tyr 122 and Arg 121 from one monomer and His 80, Arg 32 and Lys 42 from the other monomer. '''This cluster may form the active site of the breakage–reunion reaction''', with the other conserved positive charges (Arg 46 and Arg 47) anchoring the non-covalently bound 3' end of the cleaved DNA.<br /> <br /> The gyrase could be inhibited by both the quinolones and the coumarins.<br /> Coumarins prevent the B-subunit of the gyrase from hydrolyzing the ATP, so that the gyrase can't come back to its starting state for another round of supercoiling. The mecanism is not yet well known.<br /> The quinolones inhibit DNA supercoiling by gyrase, it has even the ability to induce DNA cleavage instead. It appears that these drugs will arrest any gyrase rection involving double-strand DNA breakage.<br /> However, some mutations offers quinolone resistance, most of them involved a part of the GyrA sequence : between the amino acid 67 and 106. <br /> There are also others antibiotics, like the Cinodine which binds DNA and inhibits its supercoiling by gyrase.<br /> <br /> ==See Also==<br /> *[[Gyrase|Gyrase]]<br /> *[[Topoisomerase|Topoisomerase]]<br /> <br /> ==Reference==<br /> <ref group="xtra">PMID:009278055</ref><references group="xtra"/><br /> [[Category: Escherichia coli]]<br /> [[Category: Cabral, J H.M.]]<br /> [[Category: Liddington, R C.]]<br /> [[Category: Maxwell, A.]]<br /> [[Category: Gyrase]]<br /> [[Category: Supercoiling dna]]<br /> [[Category: Topoisomerase]]<br /> [[Category: Topoisomerase ii]]<br /> *Reece, Richard J., Anthony Maxwell, and James C. Wang. "DNA gyrase: structure and function." Critical reviews in biochemistry and molecular biology 26.3-4 (1991): 335-375.</div>

Laurent Thomas https://proteopedia.org/index.php?title=Sandbox_Reserved_828&diff=1881378 Sandbox Reserved 828 2013-12-30T21:35:23Z

<p>Laurent Thomas: </p> <hr /> <div><br /> {{Sandbox_Reserved_ESBS}}<br /> <br /> <br /> ----<br /> {{STRUCTURE_1ab4| PDB=1ab4 | SCENE=56/568026/Coloured/1}} <br /> [[Image:1ab4.png|left|200px]]<br /> <br /> =='''Introduction :'''==<br /> <br /> <br /> Gyrase is the '''only prokaryote DNA topoisomerase II able to introduce negative supercoils in the DNA''' (see [http://en.wikipedia.org/wiki/DNA_supercoil DNA Supercoil]) in order to remove positive supercoils. It '''catalyses the hydrolysis of two phosphodiester bonds in a DNA segment (DNA cleavage)''' called G segment. Then, thanks to '''ATP dependant conformation changes''', it enables the passage of another segment (the T segment) through the break, and then religates the broken segment. Gyrase acts prior to the replication (before the replication fork) or other mecanisms requiring loose DNA. <br /> '''In abscence of ATP, like other topoisomerases II, gyrase only relaxes supercoils.'''<br /> <br /> Gyrase is '''coded by two differents contiguous genes gyrA and gyrB''' as it is a 350 kDa '''A<sub>2</sub>B<sub>2</sub> heterotetramers''' of two A proteins and two B proteins. <br /> The A protein breaks and religates DNA . <br /> The B protein has ATPase activity<br /> <br /> ----<br /> =='''Structure'''==<br /> <br /> [[Image:Gyrasegene.jpg]]<br /> <br /> '''The A protein''' breaks and religates DNA as the DNA cleavage core and the CTD lies on it. <br /> The gyrA 59kDa N-ter domain is called Breakage and reunion domain. It is composed of two domains at the <scene name='56/568026/Head/2'>head</scene> region :<br /> <br /> *a winged-helix-turn-helix domain or <scene name='56/568026/Whd/1'>winged helix domain (WHD</scene>) where lies the catalytic tyrosines: the active-site tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> are on loops at either end of the dimer interface.<br /> <br /> *a <scene name='56/568026/Tower/1'>tower domain</scene> with alpha/beta structure that participates in DNA bending during cleavage.<br /> <br /> The ‘head’ dimer interface is dominated by an antiparallel side by-side packing of the <scene name='56/568026/Alpha3/2'>alpha 3</scene> (the first helix of the HTH motif) from each monomer, together with their adjoining loops. At the top of the interface, the ‘recognition’ helices <scene name='56/568026/Alpha4/1'>alpha4</scene> make a head-to-head antiparallel dimer contact.<br /> <br /> <br /> There is a <scene name='56/568026/Coiledcoil_141819/1'>Coiled-coil</scene> domain, folowing the tower, with a helical core at the tail region. Two long helices (<br /> <scene name='56/568026/Helix14/1'>alpha helix 14</scene> and <scene name='56/568026/Helix18/1'>alpha helix 18</scene>) emanate from this core and connect, together with the C-terminal helix (<br /> <scene name='56/568026/Helix19/1'>alpha helix 19</scene>), the head and tail fragments. This domain has a small globular domain at it's end and is involved in the dimerization creating the <scene name='56/568026/Coiledcoil/1'>C-gate</scene> .<br /> <br /> <br /> The three connecting helices (α14,α18 and α19) adopt very different conformations, leading to large quaternary movements involving a single hinge-point within the helices and rigid body movements of <br /> the head fragments. <br /> <br /> [[Image:helixrot.jpg]] [[Image:gyrA592.jpg]]<br /> <br /> The tail is structurally conserved although large surface loops emanating from different points give it a different outward appearance. <br /> GyrA forms a heart-shaped homodimer with two protein interfaces, the DNA- and C-gates. GyrA59 is the minimal fragment of the A-subunit which, when complexed with the B-subunit, has DNA-cleavage activity. <br /> The remaining 30–35 kDa comprising the C-terminal domain (CTD) of GyrA shows a domain forming a b-pinwheel with a positively charged amino-acid perimeter. This carboxy-terminal domain of GyrA (cyan) is required for the introduction of DNA supercoils), DNA wraps around it. It is linked to the central part of Gyrase A by a flexible 13-15 residues region and stands close to the C-gate and may present an up and down movement in the early stades of the catalytic cycle and may be involved in the helping of G segment and D-gate binding so as the CTD binding of DNA.<br /> <br /> '''The B protein''' has the GHKL ATPase domain, a transducer domain and the Toprim fold on it. <br /> Two ATPase domains from two monomers dimerize to form a closed conformation, the N-gate. <br /> The Toprim fold is a Rossmann fold (beta-alpha-beta-alpha-beta) that contains three invariant acidic residues that coordinate magnesium ions involved in DNA cleavage and DNA religation. This torpim fold interacts with the Winged helix domain of gyrA to create the D-gate.<br /> <br /> <br /> '''The central core''' of the protein is made out of the association of GyrA and Gyr B with the Toprim fold, the DNA-binding domain (WHD)and tower domain, all three creating the D-gate.<br /> These domains are composed of numerous positively charged residues to interact with DNA.<br /> The Toprim domain is flexible and that this flexibility can allow the Toprim domain to coordinate with <br /> the WHD to form a competent cleavage complex.<br /> DNA is bent by ~150 degrees through an invariant isoleucine (I172)<br /> <br /> <br /> [[Image:Gyrasegates.jpg]]<br /> <br /> <br /> ----<br /> '''Mecanism'''<br /> <br /> 40-bp of duplex DNA, the G-segment, bind to the core of the enzyme and are cleaved by the active site tyrosines, while another DNA duplex, the T-segment, is captured through the ATP-induced dimerization of a protein gate, the N-gate. After passage through the transiently broken G-segment (DNA gate), the T-segment exits the protein through another protein gate, the C-gate. ATP hydrolysis and release reset the conformation of the enzyme and DNA to their initial state, poised for another strand-passage event or release of the DNA.<br /> N- and C-gates that open or close to allow T-segment transport through both theprotein and the cleaved G-segment.<br /> <br /> <br /> [[Image:mecanismgyrase.jpg]]<br /> <br /> ----<br /> <br /> =='''Reaction'''==<br /> <br /> The process of DNA supercoiling by gyrase is the following :<br /> # Gyrase binds DNA (gyrase binds more strongly to relaxed or linear DNA than to supercoiled one by a factor of about ten)<br /> # Cleavage of the G-segment<br /> # Passage of T-segment of the other strand through the cleavage site (thanks to ATP)<br /> # Reunion of the G segment<br /> # Translocation<br /> <br /> The A subunit is responsible for the breakage reunion of DNA whereas the B-subunit has the ATPase activity.<br /> The introduction of negative supercoils requires the energy of ATP-hydrolysis. 2 ATP are hydrolyzed per reaction (2 B-subunits in the gyrase)so that the linking number (Lk) changes in step of 2. The ATPase activity of the B subunit is partially located in the N-terminal region of protein B whereas the C-terminal is involved in the interaction with the A subunit and DNA. <br /> The B subunit has a weak ATPase activity in the absence of A and DNA, eventhought the A subunit is able to bind DNA without the B subunit. Both subunits are required for all the reactions of gyrase.<br /> The DNA supercoiling reaction requires in addition to ATP, a divalent cation such as Mg<sup>2+</sup>, and is stimulated in the presence of spermidine. <br /> Therefore, the gyrase could also catalyse the following reaction :<br /> * Relaxation of negative/positive supercoils<br /> * Catenation/Decatenation of closed-circular DNA<br /> * DNA cleavage (induced by Quinolone)<br /> * Unknotting DNA strands<br /> <br /> Contrary to the introduction of negative supercoils, some of these reactions don't necessarily need the energy from ATP-hydrolysis.<br /> <br /> The cleavage of DNA is achieved by a '''transesterification reaction between the tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> and the target phosphoryl groups on opposing strands of the DNA backbone''', resulting in the tyrosine being covalentlty attached to the 5' end of the cleaved segment with a 4-base overhang.<br /> The '''active site tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene>''' are on loops at either end of the dimer interface, 30Å apart, and sit at the ends of strongly basic grooves created by the dimer-related monomers.<br /> <br /> The gyrase structure reveals a '''new cluster of conserved residues''', juxtaposing Tyr 122 and Arg 121 from one monomer and His 80, Arg 32 and Lys 42 from the other monomer. '''This cluster may form the active site of the breakage–reunion reaction''', with the other conserved positive charges (Arg 46 and Arg 47) anchoring the non-covalently bound 3' end of the cleaved DNA.<br /> <br /> The gyrase could be inhibited by both the quinolones and the coumarins.<br /> Coumarins prevent the B-subunit of the gyrase from hydrolyzing the ATP, so that the gyrase can't come back to its starting state for another round of supercoiling. The mecanism is not yet well known.<br /> The quinolones inhibit DNA supercoiling by gyrase, it has even the ability to induce DNA cleavage instead. It appears that these drugs will arrest any gyrase rection involving double-strand DNA breakage.<br /> However, some mutations offers quinolone resistance, most of them involved a part of the GyrA sequence : between the amino acid 67 and 106. <br /> There are also others antibiotics, like the Cinodine which binds DNA and inhibits its supercoiling by gyrase.<br /> <br /> ==See Also==<br /> *[[Gyrase|Gyrase]]<br /> *[[Topoisomerase|Topoisomerase]]<br /> <br /> ==Reference==<br /> <ref group="xtra">PMID:009278055</ref><references group="xtra"/><br /> [[Category: Escherichia coli]]<br /> [[Category: Cabral, J H.M.]]<br /> [[Category: Liddington, R C.]]<br /> [[Category: Maxwell, A.]]<br /> [[Category: Gyrase]]<br /> [[Category: Supercoiling dna]]<br /> [[Category: Topoisomerase]]<br /> [[Category: Topoisomerase ii]]<br /> *Reece, Richard J., Anthony Maxwell, and James C. Wang. "DNA gyrase: structure and function." Critical reviews in biochemistry and molecular biology 26.3-4 (1991): 335-375.</div>

Laurent Thomas https://proteopedia.org/index.php?title=Sandbox_Reserved_828&diff=1881377 Sandbox Reserved 828 2013-12-30T21:25:33Z

<p>Laurent Thomas: </p> <hr /> <div><br /> {{Sandbox_Reserved_ESBS}}<br /> <br /> <br /> ----<br /> {{STRUCTURE_1ab4| PDB=1ab4 | SCENE=56/568026/Coloured/1}} <br /> [[Image:1ab4.png|left|200px]]<br /> <br /> =='''Introduction :'''==<br /> <br /> <br /> Gyrase is the '''only prokaryote DNA topoisomerase II able to introduce negative supercoils in the DNA''' (see [http://en.wikipedia.org/wiki/DNA_supercoil DNA Supercoil]) in order to remove positive supercoils. It '''catalyses the hydrolysis of two phosphodiester bonds in a DNA segment (DNA cleavage)''' called G segmen. Then, thanks to '''ATP dependant conformation changes''' it enables the passage of another segment (the T segment) through the break, and then religates the broken segment. Gyrase acts prior to the replication (before the replication fork) or other mecanisms requiring loose DNA. <br /> '''In abscence of ATP, like other topoisomerases II, gyrase only relaxes supercoils.'''<br /> <br /> Gyrase is coded by two differents contiguous genes gyrA and GgyrB as it is a 350 kDa A<sub>2</sub>B<sub>2</sub> heterotetramers of two A proteins and two B proteins. <br /> The A protein breaks and religates DNA . The B protein has ATPase activity<br /> <br /> ----<br /> =='''Structure'''==<br /> <br /> [[Image:Gyrasegene.jpg]]<br /> <br /> '''The A protein''' breaks and religates DNA as the DNA cleavage core and the CTD lies on it. <br /> The gyrA 59kDa N-ter domain is called Breakage and reunion domain. It is composed of two domains at the <scene name='56/568026/Head/2'>head</scene> region :<br /> <br /> *a winged-helix-turn-helix domain or <scene name='56/568026/Whd/1'>winged helix domain (WHD</scene>) where lies the catalytic tyrosines: the active-site tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> are on loops at either end of the dimer interface.<br /> <br /> *a <scene name='56/568026/Tower/1'>tower domain</scene> with alpha/beta structure that participates in DNA bending during cleavage.<br /> <br /> The ‘head’ dimer interface is dominated by an antiparallel side by-side packing of the <scene name='56/568026/Alpha3/2'>alpha 3</scene> (the first helix of the HTH motif) from each monomer, together with their adjoining loops. At the top of the interface, the ‘recognition’ helices <scene name='56/568026/Alpha4/1'>alpha4</scene> make a head-to-head antiparallel dimer contact.<br /> <br /> <br /> There is a <scene name='56/568026/Coiledcoil_141819/1'>Coiled-coil</scene> domain, folowing the tower, with a helical core at the tail region. Two long helices (<br /> <scene name='56/568026/Helix14/1'>alpha helix 14</scene> and <scene name='56/568026/Helix18/1'>alpha helix 18</scene>) emanate from this core and connect, together with the C-terminal helix (<br /> <scene name='56/568026/Helix19/1'>alpha helix 19</scene>), the head and tail fragments. This domain has a small globular domain at it's end and is involved in the dimerization creating the <scene name='56/568026/Coiledcoil/1'>C-gate</scene> .<br /> <br /> <br /> The three connecting helices (α14,α18 and α19) adopt very different conformations, leading to large quaternary movements involving a single hinge-point within the helices and rigid body movements of <br /> the head fragments. <br /> <br /> [[Image:helixrot.jpg]] [[Image:gyrA592.jpg]]<br /> <br /> The tail is structurally conserved although large surface loops emanating from different points give it a different outward appearance. <br /> GyrA forms a heart-shaped homodimer with two protein interfaces, the DNA- and C-gates. GyrA59 is the minimal fragment of the A-subunit which, when complexed with the B-subunit, has DNA-cleavage activity. <br /> The remaining 30–35 kDa comprising the C-terminal domain (CTD) of GyrA shows a domain forming a b-pinwheel with a positively charged amino-acid perimeter. This carboxy-terminal domain of GyrA (cyan) is required for the introduction of DNA supercoils), DNA wraps around it. It is linked to the central part of Gyrase A by a flexible 13-15 residues region and stands close to the C-gate and may present an up and down movement in the early stades of the catalytic cycle and may be involved in the helping of G segment and D-gate binding so as the CTD binding of DNA.<br /> <br /> '''The B protein''' has the GHKL ATPase domain, a transducer domain and the Toprim fold on it. <br /> Two ATPase domains from two monomers dimerize to form a closed conformation, the N-gate. <br /> The Toprim fold is a Rossmann fold (beta-alpha-beta-alpha-beta) that contains three invariant acidic residues that coordinate magnesium ions involved in DNA cleavage and DNA religation. This torpim fold interacts with the Winged helix domain of gyrA to create the D-gate.<br /> <br /> <br /> '''The central core''' of the protein is made out of the association of GyrA and Gyr B with the Toprim fold, the DNA-binding domain (WHD)and tower domain, all three creating the D-gate.<br /> These domains are composed of numerous positively charged residues to interact with DNA.<br /> The Toprim domain is flexible and that this flexibility can allow the Toprim domain to coordinate with <br /> the WHD to form a competent cleavage complex.<br /> DNA is bent by ~150 degrees through an invariant isoleucine (I172)<br /> <br /> <br /> [[Image:Gyrasegates.jpg]]<br /> <br /> <br /> ----<br /> '''Mecanism'''<br /> <br /> 40-bp of duplex DNA, the G-segment, bind to the core of the enzyme and are cleaved by the active site tyrosines, while another DNA duplex, the T-segment, is captured through the ATP-induced dimerization of a protein gate, the N-gate. After passage through the transiently broken G-segment (DNA gate), the T-segment exits the protein through another protein gate, the C-gate. ATP hydrolysis and release reset the conformation of the enzyme and DNA to their initial state, poised for another strand-passage event or release of the DNA.<br /> N- and C-gates that open or close to allow T-segment transport through both theprotein and the cleaved G-segment.<br /> <br /> <br /> [[Image:mecanismgyrase.jpg]]<br /> <br /> ----<br /> <br /> =='''Reaction'''==<br /> <br /> The process of DNA supercoiling by gyrase is the following :<br /> # Gyrase binds DNA (gyrase binds more strongly to relaxed or linear DNA than to supercoiled one by a factor of about ten)<br /> # Cleavage of the G-segment<br /> # Passage of T-segment of the other strand through the cleavage site (thanks to ATP)<br /> # Reunion of the G segment<br /> # Translocation<br /> <br /> The A subunit is responsible for the breakage reunion of DNA whereas the B-subunit has the ATPase activity.<br /> The introduction of negative supercoils requires the energy of ATP-hydrolysis. 2 ATP are hydrolyzed per reaction (2 B-subunits in the gyrase)so that the linking number (Lk) changes in step of 2. The ATPase activity of the B subunit is partially located in the N-terminal region of protein B whereas the C-terminal is involved in the interaction with the A subunit and DNA. <br /> The B subunit has a weak ATPase activity in the absence of A and DNA, eventhought the A subunit is able to bind DNA without the B subunit. Both subunits are required for all the reactions of gyrase.<br /> The DNA supercoiling reaction requires in addition to ATP, a divalent cation such as Mg<sup>2+</sup>, and is stimulated in the presence of spermidine. <br /> Therefore, the gyrase could also catalyse the following reaction :<br /> * Relaxation of negative/positive supercoils<br /> * Catenation/Decatenation of closed-circular DNA<br /> * DNA cleavage (induced by Quinolone)<br /> * Unknotting DNA strands<br /> <br /> Contrary to the introduction of negative supercoils, some of these reactions don't necessarily need the energy from ATP-hydrolysis.<br /> <br /> The cleavage of DNA is achieved by a '''transesterification reaction between the tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> and the target phosphoryl groups on opposing strands of the DNA backbone''', resulting in the tyrosine being covalentlty attached to the 5' end of the cleaved segment with a 4-base overhang.<br /> The '''active site tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene>''' are on loops at either end of the dimer interface, 30Å apart, and sit at the ends of strongly basic grooves created by the dimer-related monomers.<br /> <br /> The gyrase structure reveals a '''new cluster of conserved residues''', juxtaposing Tyr 122 and Arg 121 from one monomer and His 80, Arg 32 and Lys 42 from the other monomer. '''This cluster may form the active site of the breakage–reunion reaction''', with the other conserved positive charges (Arg 46 and Arg 47) anchoring the non-covalently bound 3' end of the cleaved DNA.<br /> <br /> The gyrase could be inhibited by both the quinolones and the coumarins.<br /> Coumarins prevent the B-subunit of the gyrase from hydrolyzing the ATP, so that the gyrase can't come back to its starting state for another round of supercoiling. The mecanism is not yet well known.<br /> The quinolones inhibit DNA supercoiling by gyrase, it has even the ability to induce DNA cleavage instead. It appears that these drugs will arrest any gyrase rection involving double-strand DNA breakage.<br /> However, some mutations offers quinolone resistance, most of them involved a part of the GyrA sequence : between the amino acid 67 and 106. <br /> There are also others antibiotics, like the Cinodine which binds DNA and inhibits its supercoiling by gyrase.<br /> <br /> ==See Also==<br /> *[[Gyrase|Gyrase]]<br /> *[[Topoisomerase|Topoisomerase]]<br /> <br /> ==Reference==<br /> <ref group="xtra">PMID:009278055</ref><references group="xtra"/><br /> [[Category: Escherichia coli]]<br /> [[Category: Cabral, J H.M.]]<br /> [[Category: Liddington, R C.]]<br /> [[Category: Maxwell, A.]]<br /> [[Category: Gyrase]]<br /> [[Category: Supercoiling dna]]<br /> [[Category: Topoisomerase]]<br /> [[Category: Topoisomerase ii]]<br /> *Reece, Richard J., Anthony Maxwell, and James C. Wang. "DNA gyrase: structure and function." Critical reviews in biochemistry and molecular biology 26.3-4 (1991): 335-375.</div>

Laurent Thomas https://proteopedia.org/index.php?title=Sandbox_Reserved_828&diff=1881260 Sandbox Reserved 828 2013-12-29T22:43:23Z

<p>Laurent Thomas: </p> <hr /> <div><br /> {{Sandbox_Reserved_ESBS}}<br /> <br /> <br /> ----<br /> {{STRUCTURE_1ab4| PDB=1ab4 | SCENE=56/568026/Coloured/1}} <br /> [[Image:1ab4.png|left|200px]]<br /> <br /> =='''Introduction :'''==<br /> <br /> <br /> Gyrase is the '''only prokaryote DNA topoisomerase II able to introduce negative supercoils in the DNA''' (see [http://en.wikipedia.org/wiki/DNA_supercoil DNA Supercoil]) in order to remove positive supercoils. It '''catalyses the hydrolysis of two phosphodiester bonds in a DNA segment (DNA cleavage)''' (called G segment). Then, thanks to '''ATP dependant conformation changes''' it enables the passage of another segment (the T segment) through the break, and then religates the broken segment. Gyrase acts prior to the replication (before the replication fork) or other mecanisms requiring loose DNA. <br /> '''In abscence of ATP, like other topoisomerases II, gyrase only relaxes supercoils.'''<br /> <br /> Gyrase is coded by two differents contiguous genes gyrA and GgyrB as it is a 350 kDa A<sub>2</sub>B<sub>2</sub> heterotetramers of two A proteins and two B proteins. <br /> The A protein breaks and religates DNA . The B protein has ATPase activity<br /> <br /> ----<br /> =='''Structure'''==<br /> <br /> [[Image:Gyrasegene.jpg]]<br /> <br /> '''The A protein''' breaks and religates DNA as the DNA cleavage core and the CTD lies on it. <br /> The gyrA 59kDa N-ter domain is called Breakage and reunion domain. It is composed of two domains at the <scene name='56/568026/Head/2'>head</scene> region :<br /> <br /> a winged-helix-turn-helix domain or <scene name='56/568026/Whd/1'>winged helix domain (WHD</scene>) where lies the catalytic tyrosines: the active-site tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> are on loops at either end of the dimer interface.<br /> <br /> a <scene name='56/568026/Tower/1'>tower domain</scene> with alpha/beta structure that participates in DNA bending during cleavage.<br /> <br /> The ‘head’ dimer interface is dominated by an antiparallel side by-side packing of the <scene name='56/568026/Alpha3/2'>alpha 3</scene> (the first helix of the HTH motif) from each monomer, together with their adjoining loops. At the top of the interface, the ‘recognition’ helices <scene name='56/568026/Alpha4/1'>alpha4</scene> make a head-to-head antiparallel dimer contact.<br /> <br /> <br /> There is a <scene name='56/568026/Coiledcoil_141819/1'>Coiled-coil</scene> domain, folowing the tower, with a helical core at the tail region. Two long helices (<br /> <scene name='56/568026/Helix14/1'>alpha helix 14</scene> and <scene name='56/568026/Helix18/1'>alpha helix 18</scene>) emanate from this core and connect, together with the C-terminal helix (<br /> <scene name='56/568026/Helix19/1'>alpha helix 19</scene>), the head and tail fragments. This domain has a small globular domain at it's end and is involved in the dimerization creating the <scene name='56/568026/Coiledcoil/1'>C-gate</scene> .<br /> <br /> <br /> The three connecting helices (α14,α18 and α19) adopt very different conformations, leading to large quaternary movements involving a single hinge-point within the helices and rigid body movements of <br /> the head fragments. <br /> <br /> [[Image:helixrot.jpg]] [[Image:gyrA592.jpg]]<br /> <br /> The tail is structurally conserved although large surface loops emanating from different points give it a different outward appearance. <br /> GyrA forms a heart-shaped homodimer with two protein interfaces, the DNA- and C-gates. GyrA59 is the minimal fragment of the A-subunit which, when complexed with the B-subunit, has DNA-cleavage activity. <br /> The remaining 30–35 kDa comprising the C-terminal domain (CTD) of GyrA shows a domain forming a b-pinwheel with a positively charged amino-acid perimeter. This carboxy-terminal domain of GyrA (cyan) is required for the introduction of DNA supercoils), DNA wraps around it. It is linked to the central part of Gyrase A by a flexible 13-15 residues region and stands close to the C-gate and may present an up and down movement in the early stades of the catalytic cycle and may be involved in the helping of G segment and D-gate binding so as the CTD binding of DNA.<br /> <br /> '''The B protein''' has the GHKL ATPase domain, a transducer domain and the Toprim fold on it. <br /> Two ATPase domains from two monomers dimerize to form a closed conformation, the N-gate. <br /> The Toprim fold is a Rossmann fold (beta-alpha-beta-alpha-beta) that contains three invariant acidic residues that coordinate magnesium ions involved in DNA cleavage and DNA religation. This torpim fold interacts with the Winged helix domain of gyrA to create the D-gate.<br /> <br /> <br /> '''The central core''' of the protein is made out of the association of GyrA and Gyr B with the Toprim fold, the DNA-binding domain (WHD)and tower domain, all three creating the D-gate.<br /> These domains are composed of numerous positively charged residues to interact with DNA.<br /> The Toprim domain is flexible and that this flexibility can allow the Toprim domain to coordinate with <br /> the WHD to form a competent cleavage complex.<br /> DNA is bent by ~150 degrees through an invariant isoleucine (I172)<br /> <br /> <br /> [[Image:Gyrasegates.jpg]]<br /> <br /> <br /> ----<br /> '''Mecanism'''<br /> <br /> 40-bp of duplex DNA, the G-segment, bind to the core of the enzyme and are cleaved by the active site tyrosines, while another DNA duplex, the T-segment, is captured through the ATP-induced dimerization of a protein gate, the N-gate. After passage through the transiently broken G-segment (DNA gate), the T-segment exits the protein through another protein gate, the C-gate. ATP hydrolysis and release reset the conformation of the enzyme and DNA to their initial state, poised for another strand-passage event or release of the DNA.<br /> N- and C-gates that open or close to allow T-segment transport through both theprotein and the cleaved G-segment.<br /> <br /> <br /> [[Image:mecanismgyrase.jpg]]<br /> <br /> ----<br /> <br /> =='''Reaction'''==<br /> <br /> The process of DNA supercoiling by gyrase is the following :<br /> # Gyrase binds DNA (gyrase binds more strongly to relaxed or linear DNA than to supercoiled one by a factor of about ten)<br /> # Cleavage of the G-segment<br /> # Passage of T-segment of the other strand through the cleavage site (thanks to DNA)<br /> # Reunion of the G segment<br /> # Translocation<br /> <br /> The A subunit is responsible for the breakage reunion of DNA whereas the B-subunit has the ATPase activity.<br /> The introduction of negative supercoils requires the energy of ATP-hydrolysis. 2 ATP are hydrolyzed per reaction (2 B-subunits in the gyrase)so that the linking number (Lk) changes in step of 2. The ATPase activity of the B subunit is partially located in the N-terminal region of protein B whereas the C-terminal is involved in the interaction with the A subunit and DNA. <br /> The B subunit has a weak ATPase activity in the absence of A and DNA, eventhought the A subunit is able to bind DNA without the B subunit. Both subunits are required for all the reactions of gyrase.<br /> The DNA supercoiling reaction requires in addition to ATP, a divalent cation such as Mg<sup>2+</sup>, and is stimulated in the presence of spermidine. <br /> Therefore, the gyrase could also catalyse the following reaction :<br /> * Relaxation of negative/positive supercoils<br /> * Catenation/Decatenation of closed-circular DNA<br /> * DNA cleavage (induced by Quinolone)<br /> * Unknotting DNA strands<br /> <br /> Contrary to the introduction of negative supercoils, some of these reactions don't necessarily need the energy from ATP-hydrolysis.<br /> <br /> The cleavage of DNA is achieved by a '''transesterification reaction between the tyrosines (Tyr122) and the target phosphoryl groups on opposing strands of the DNA backbone''', resulting in the tyrosine being covalentlty attached to the 5' end of the cleaved segment with a 4-base overhang.<br /> The '''active site tyrosines (Tyr 122)''' are on loops at either end of the dimer interface, 30Å apart, and sit at the ends of strongly basic grooves created by the dimer-related monomers.<br /> <br /> The gyrase structure reveals a '''new cluster of conserved residues''', juxtaposing Tyr 122 and Arg 121 from one monomer and His 80, Arg 32 and Lys 42 from the other monomer. '''This cluster may form the active site of the breakage–reunion reaction''', with the other conserved positive charges (Arg 46 and Arg 47) anchoring the non-covalently bound 3' end of the cleaved DNA.<br /> <br /> The gyrase could be inhibited by both the quinolones and the coumarins.<br /> Coumarins prevent the B-subunit of the gyrase from hydrolyzing the ATP, so that the gyrase can't come back to its starting state for another round of supercoiling. The mecanism is not yet well known.<br /> The quinolones inhibit DNA supercoiling by gyrase, it has even the ability to induce DNA cleavage instead. It appears that these drugs will arrest any gyrase rection involving double-strand DNA breakage.<br /> However, some mutations offers quinolone resistance, most of them involved a part of the GyrA sequence : between the amino acid 67 and 106. <br /> There are also others antibiotics, like the Cinodine which binds DNA and inhibits its supercoiling by gyrase.<br /> <br /> ==See Also==<br /> *[[Gyrase|Gyrase]]<br /> <br /> ==Reference==<br /> <ref group="xtra">PMID:009278055</ref><references group="xtra"/><br /> [[Category: Escherichia coli]]<br /> [[Category: Cabral, J H.M.]]<br /> [[Category: Liddington, R C.]]<br /> [[Category: Maxwell, A.]]<br /> [[Category: Gyrase]]<br /> [[Category: Supercoiling dna]]<br /> [[Category: Topoisomerase]]<br /> [[Category: Topoisomerase ii]]<br /> *Reece, Richard J., Anthony Maxwell, and James C. Wang. "DNA gyrase: structure and function." Critical reviews in biochemistry and molecular biology 26.3-4 (1991): 335-375.</div>

Laurent Thomas https://proteopedia.org/index.php?title=Sandbox_Reserved_828&diff=1881259 Sandbox Reserved 828 2013-12-29T22:35:11Z

<p>Laurent Thomas: </p> <hr /> <div><br /> {{Sandbox_Reserved_ESBS}}<br /> <br /> <br /> ----<br /> {{STRUCTURE_1ab4| PDB=1ab4 | SCENE=56/568026/Coloured/1}} <br /> [[Image:1ab4.png|left|200px]]<br /> <br /> =='''Introduction :'''==<br /> <br /> <br /> Gyrase is the '''only prokaryote DNA topoisomerase II able to introduce negative supercoils in the DNA''' (see [http://en.wikipedia.org/wiki/DNA_supercoil DNA Supercoil]) in order to remove positive supercoils. It '''catalyses the hydrolysis of two phosphodiester bonds in a DNA segment (DNA cleavage)''' (called G segment). Then, thanks to '''ATP dependant conformation changes''' it enables the passage of another segment (the T segment) through the break, and then religates the broken segment. Gyrase acts prior to the replication (before the replication fork) or other mecanisms requiring loose DNA. <br /> '''In abscence of ATP, like other topoisomerases II, gyrase only relaxes supercoils.'''<br /> <br /> Gyrase is coded by two differents contiguous genes gyrA and GgyrB as it is a 350 kDa A<sub>2</sub>B<sub>2</sub> heterotetramers of two A proteins and two B proteins. <br /> The A protein breaks and religates DNA . The B protein has ATPase activity<br /> <br /> ----<br /> =='''Structure'''==<br /> <br /> [[Image:Gyrasegene.jpg]]<br /> <br /> '''The A protein''' breaks and religates DNA as the DNA cleavage core and the CTD lies on it. <br /> The gyrA 59kDa N-ter domain is called Breakage and reunion domain. It is composed of two domains at the <scene name='56/568026/Head/2'>head</scene> region :<br /> <br /> a winged-helix-turn-helix domain or <scene name='56/568026/Whd/1'>winged helix domain (WHD</scene>) where lies the catalytic tyrosines: the active-site tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> are on loops at either end of the dimer interface.<br /> <br /> a <scene name='56/568026/Tower/1'>tower domain</scene> with alpha/beta structure that participates in DNA bending during cleavage.<br /> <br /> The ‘head’ dimer interface is dominated by an antiparallel side by-side packing of the <scene name='56/568026/Alpha3/2'>alpha 3</scene> (the first helix of the HTH motif) from each monomer, together with their adjoining loops. At the top of the interface, the ‘recognition’ helices <scene name='56/568026/Alpha4/1'>alpha4</scene> make a head-to-head antiparallel dimer contact.<br /> <br /> <br /> There is a <scene name='56/568026/Coiledcoil_141819/1'>Coiled-coil</scene> domain, folowing the tower, with a helical core at the tail region. Two long helices (<br /> <scene name='56/568026/Helix14/1'>alpha helix 14</scene> and <scene name='56/568026/Helix18/1'>alpha helix 18</scene>) emanate from this core and connect, together with the C-terminal helix (<br /> <scene name='56/568026/Helix19/1'>alpha helix 19</scene>), the head and tail fragments. This domain has a small globular domain at it's end and is involved in the dimerization creating the <scene name='56/568026/Coiledcoil/1'>C-gate</scene> .<br /> <br /> <br /> The three connecting helices (α14,α18 and α19) adopt very different conformations, leading to large quaternary movements involving a single hinge-point within the helices and rigid body movements of <br /> the head fragments. <br /> <br /> [[Image:helixrot.jpg]] [[Image:gyrA592.jpg]]<br /> <br /> The tail is structurally conserved although large surface loops emanating from different points give it a different outward appearance. <br /> GyrA forms a heart-shaped homodimer with two protein interfaces, the DNA- and C-gates. GyrA59 is the minimal fragment of the A-subunit which, when complexed with the B-subunit, has DNA-cleavage activity. <br /> The remaining 30–35 kDa comprising the C-terminal domain (CTD) of GyrA shows a domain forming a b-pinwheel with a positively charged amino-acid perimeter. This carboxy-terminal domain of GyrA (cyan) is required for the introduction of DNA supercoils), DNA wraps around it. It is linked to the central part of Gyrase A by a flexible 13-15 residues region and stands close to the C-gate and may present an up and down movement in the early stades of the catalytic cycle and may be involved in the helping of G segment and D-gate binding so as the CTD binding of DNA.<br /> <br /> '''The B protein''' has the GHKL ATPase domain, a transducer domain and the Toprim fold on it. <br /> Two ATPase domains from two monomers dimerize to form a closed conformation, the N-gate. <br /> The Toprim fold is a Rossmann fold (beta-alpha-beta-alpha-beta) that contains three invariant acidic residues that coordinate magnesium ions involved in DNA cleavage and DNA religation. This torpim fold interacts with the Winged helix domain of gyrA to create the D-gate.<br /> <br /> <br /> '''The central core''' of the protein is made out of the association of GyrA and Gyr B with the Toprim fold, the DNA-binding domain (WHD)and tower domain, all three creating the D-gate.<br /> These domains are composed of numerous positively charged residues to interact with DNA.<br /> The Toprim domain is flexible and that this flexibility can allow the Toprim domain to coordinate with <br /> the WHD to form a competent cleavage complex.<br /> DNA is bent by ~150 degrees through an invariant isoleucine (I172)<br /> <br /> <br /> [[Image:Gyrasegates.jpg]]<br /> <br /> <br /> ----<br /> '''Mecanism'''<br /> <br /> 40-bp of duplex DNA, the G-segment, bind to the core of the enzyme and are cleaved by the active site tyrosines, while another DNA duplex, the T-segment, is captured through the ATP-induced dimerization of a protein gate, the N-gate. After passage through the transiently broken G-segment (DNA gate), the T-segment exits the protein through another protein gate, the C-gate. ATP hydrolysis and release reset the conformation of the enzyme and DNA to their initial state, poised for another strand-passage event or release of the DNA.<br /> N- and C-gates that open or close to allow T-segment transport through both theprotein and the cleaved G-segment.<br /> <br /> <br /> [[Image:mecanismgyrase.jpg]]<br /> <br /> ----<br /> <br /> =='''Reaction'''==<br /> <br /> The process of DNA supercoiling by gyrase is the following :<br /> # gyrase binds DNA (gyrase binds more strongly to relaxed or linear DNA than to supercoiled one by a factor of about ten)<br /> # cleavage of the G-segment<br /> # passage of T-segment of the other strand through the cleavage site (thanks to DNA)<br /> # Reunion of the G segment<br /> # Translocation<br /> <br /> The A subunit is responsible for the breakage reunion of DNA whereas the B-subunit has the ATPase activity.<br /> The introduction of negative supercoils requires the energy of ATP-hydrolysis. 2 ATP are hydrolyzed per reaction (2 B-subunits in the gyrase)so that the linking number (Lk) changes in step of 2. The ATPase activity of the B subunit is partially located in the N-terminal region of protein B whereas the C-terminal is involved in the interaction with the A subunit and DNA. <br /> The B subunit has a weak ATPase activity in the absence of A and DNA, eventhought the A subunit is able to bind DNA without the B subunit. Both subunits are required for all the reactions of gyrase.<br /> The DNA supercoiling reaction requires in addition to ATP, a divalent cation such as Mg<sup>2+</sup>, and is stimulated in the presence of spermidine. <br /> Therefore, the gyrase could also catalyse the following reaction :<br /> * Relaxation of negative/positive supercoils<br /> * Catenation/Decatenation of closed-circular DNA<br /> * DNA cleavage (induced by Quinolone)<br /> * Unknotting DNA strands<br /> <br /> Contrary to the introduction of negative supercoils, some of these reactions don't necessarily need the energy from ATP-hydrolysis.<br /> <br /> The cleavage of DNA is achieved by a '''transesterification reaction between the tyrosines (Tyr122) and the target phosphoryl groups on opposing strands of the DNA backbone''', resulting in the tyrosine being covalentlty attached to the 5' end of the cleaved segment with a 4-base overhang.<br /> The '''active site tyrosines (Tyr 122)''' are on loops at either end of the dimer interface, 30Å apart, and sit at the ends of strongly basic grooves created by the dimer-related monomers.<br /> <br /> The gyrase structure reveals a '''new cluster of conserved residues''', juxtaposing Tyr 122 and Arg 121 from one monomer and His 80, Arg 32 and Lys 42 from the other monomer. '''This cluster may form the active site of the breakage–reunion reaction''', with the other conserved positive charges (Arg 46 and Arg 47) anchoring the non-covalently bound 3' end of the cleaved DNA.<br /> <br /> The gyrase could be inhibited by both the quinolones and the coumarins.<br /> Coumarins prevent the B-subunit of the gyrase from hydrolyzing the ATP, so that the gyrase can't come back to its starting state for another round of supercoiling. The mecanism is not yet well known.<br /> The quinolones inhibit DNA supercoiling by gyrase, it has even the ability to induce DNA cleavage instead. It appears that these drugs will arrest any gyrase rection involving double-strand DNA breakage.<br /> However, some mutations offers quinolone resistance, most of them involved a part of the GyrA sequence : between the amino acid 67 and 106. <br /> There are also others antibiotics, like the Cinodine which binds DNA and inhibits its supercoiling by gyrase.<br /> <br /> ==See Also==<br /> *[[Gyrase|Gyrase]]<br /> <br /> ==Reference==<br /> <ref group="xtra">PMID:009278055</ref><references group="xtra"/><br /> [[Category: Escherichia coli]]<br /> [[Category: Cabral, J H.M.]]<br /> [[Category: Liddington, R C.]]<br /> [[Category: Maxwell, A.]]<br /> [[Category: Gyrase]]<br /> [[Category: Supercoiling dna]]<br /> [[Category: Topoisomerase]]<br /> [[Category: Topoisomerase ii]]<br /> *Reece, Richard J., Anthony Maxwell, and James C. Wang. "DNA gyrase: structure and function." Critical reviews in biochemistry and molecular biology 26.3-4 (1991): 335-375.</div>

Laurent Thomas https://proteopedia.org/index.php?title=Sandbox_Reserved_828&diff=1881258 Sandbox Reserved 828 2013-12-29T22:31:12Z

<p>Laurent Thomas: </p> <hr /> <div><br /> {{Sandbox_Reserved_ESBS}}<br /> <br /> <br /> ----<br /> <Structure load='1ab4_mm1.pdb' size='400' frame='true' align='right' caption='Monomer of GyrA59 subunit at 2.8Å (1ab4.pdb)' scene='56/568026/Coloured/1'><br /> [[Image:1ab4.png|left|200px]]<br /> <br /> =='''Introduction :'''==<br /> <br /> <br /> Gyrase is the '''only prokaryote DNA topoisomerase II able to introduce negative supercoils in the DNA''' (see [http://en.wikipedia.org/wiki/DNA_supercoil DNA Supercoil]) in order to remove positive supercoils. It '''catalyses the hydrolysis of two phosphodiester bonds in a DNA segment (DNA cleavage)''' (called G segment). Then, thanks to '''ATP dependant conformation changes''' it enables the passage of another segment (the T segment) through the break, and then religates the broken segment. Gyrase acts prior to the replication (before the replication fork) or other mecanisms requiring loose DNA. <br /> '''In abscence of ATP, like other topoisomerases II, gyrase only relaxes supercoils.'''<br /> <br /> Gyrase is coded by two differents contiguous genes gyrA and GgyrB as it is a 350 kDa A<sub>2</sub>B<sub>2</sub> heterotetramers of two A proteins and two B proteins. <br /> The A protein breaks and religates DNA . The B protein has ATPase activity<br /> <br /> ----<br /> =='''Structure'''==<br /> <br /> [[Image:Gyrasegene.jpg]]<br /> <br /> '''The A protein''' breaks and religates DNA as the DNA cleavage core and the CTD lies on it. <br /> The gyrA 59kDa N-ter domain is called Breakage and reunion domain. It is composed of two domains at the <scene name='56/568026/Head/2'>head</scene> region :<br /> <br /> a winged-helix-turn-helix domain or <scene name='56/568026/Whd/1'>winged helix domain (WHD</scene>) where lies the catalytic tyrosines: the active-site tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> are on loops at either end of the dimer interface.<br /> <br /> a <scene name='56/568026/Tower/1'>tower domain</scene> with alpha/beta structure that participates in DNA bending during cleavage.<br /> <br /> The ‘head’ dimer interface is dominated by an antiparallel side by-side packing of the <scene name='56/568026/Alpha3/2'>alpha 3</scene> (the first helix of the HTH motif) from each monomer, together with their adjoining loops. At the top of the interface, the ‘recognition’ helices <scene name='56/568026/Alpha4/1'>alpha4</scene> make a head-to-head antiparallel dimer contact.<br /> <br /> <br /> There is a <scene name='56/568026/Coiledcoil_141819/1'>Coiled-coil</scene> domain, folowing the tower, with a helical core at the tail region. Two long helices (<br /> <scene name='56/568026/Helix14/1'>alpha helix 14</scene> and <scene name='56/568026/Helix18/1'>alpha helix 18</scene>) emanate from this core and connect, together with the C-terminal helix (<br /> <scene name='56/568026/Helix19/1'>alpha helix 19</scene>), the head and tail fragments. This domain has a small globular domain at it's end and is involved in the dimerization creating the <scene name='56/568026/Coiledcoil/1'>C-gate</scene> .<br /> <br /> <br /> The three connecting helices (α14,α18 and α19) adopt very different conformations, leading to large quaternary movements involving a single hinge-point within the helices and rigid body movements of <br /> the head fragments. <br /> <br /> [[Image:helixrot.jpg]] [[Image:gyrA592.jpg]]<br /> <br /> The tail is structurally conserved although large surface loops emanating from different points give it a different outward appearance. <br /> GyrA forms a heart-shaped homodimer with two protein interfaces, the DNA- and C-gates. GyrA59 is the minimal fragment of the A-subunit which, when complexed with the B-subunit, has DNA-cleavage activity. <br /> The remaining 30–35 kDa comprising the C-terminal domain (CTD) of GyrA shows a domain forming a b-pinwheel with a positively charged amino-acid perimeter. This carboxy-terminal domain of GyrA (cyan) is required for the introduction of DNA supercoils), DNA wraps around it. It is linked to the central part of Gyrase A by a flexible 13-15 residues region and stands close to the C-gate and may present an up and down movement in the early stades of the catalytic cycle and may be involved in the helping of G segment and D-gate binding so as the CTD binding of DNA.<br /> <br /> '''The B protein''' has the GHKL ATPase domain, a transducer domain and the Toprim fold on it. <br /> Two ATPase domains from two monomers dimerize to form a closed conformation, the N-gate. <br /> The Toprim fold is a Rossmann fold (beta-alpha-beta-alpha-beta) that contains three invariant acidic residues that coordinate magnesium ions involved in DNA cleavage and DNA religation. This torpim fold interacts with the Winged helix domain of gyrA to create the D-gate.<br /> <br /> <br /> '''The central core''' of the protein is made out of the association of GyrA and Gyr B with the Toprim fold, the DNA-binding domain (WHD)and tower domain, all three creating the D-gate.<br /> These domains are composed of numerous positively charged residues to interact with DNA.<br /> The Toprim domain is flexible and that this flexibility can allow the Toprim domain to coordinate with <br /> the WHD to form a competent cleavage complex.<br /> DNA is bent by ~150 degrees through an invariant isoleucine (I172)<br /> <br /> <br /> [[Image:Gyrasegates.jpg]]<br /> <br /> <br /> ----<br /> '''Mecanism'''<br /> <br /> 40-bp of duplex DNA, the G-segment, bind to the core of the enzyme and are cleaved by the active site tyrosines, while another DNA duplex, the T-segment, is captured through the ATP-induced dimerization of a protein gate, the N-gate. After passage through the transiently broken G-segment (DNA gate), the T-segment exits the protein through another protein gate, the C-gate. ATP hydrolysis and release reset the conformation of the enzyme and DNA to their initial state, poised for another strand-passage event or release of the DNA.<br /> N- and C-gates that open or close to allow T-segment transport through both theprotein and the cleaved G-segment.<br /> <br /> <br /> [[Image:mecanismgyrase.jpg]]<br /> <br /> ----<br /> <br /> =='''Reaction'''==<br /> <br /> The process of DNA supercoiling by gyrase is the following :<br /> # gyrase binds DNA (gyrase binds more strongly to relaxed or linear DNA than to supercoiled one by a factor of about ten)<br /> # cleavage of the G-segment<br /> # passage of T-segment of the other strand through the cleavage site (thanks to DNA)<br /> # Reunion of the G segment<br /> # Translocation<br /> <br /> The A subunit is responsible for the breakage reunion of DNA whereas the B-subunit has the ATPase activity.<br /> The introduction of negative supercoils requires the energy of ATP-hydrolysis. 2 ATP are hydrolyzed per reaction (2 B-subunits in the gyrase)so that the linking number (Lk) changes in step of 2. The ATPase activity of the B subunit is partially located in the N-terminal region of protein B whereas the C-terminal is involved in the interaction with the A subunit and DNA. <br /> The B subunit has a weak ATPase activity in the absence of A and DNA, eventhought the A subunit is able to bind DNA without the B subunit. Both subunits are required for all the reactions of gyrase.<br /> The DNA supercoiling reaction requires in addition to ATP, a divalent cation such as Mg<sup>2+</sup>, and is stimulated in the presence of spermidine. <br /> Therefore, the gyrase could also catalyse the following reaction :<br /> * Relaxation of negative/positive supercoils<br /> * Catenation/Decatenation of closed-circular DNA<br /> * DNA cleavage (induced by Quinolone)<br /> * Unknotting DNA strands<br /> <br /> Contrary to the introduction of negative supercoils, some of these reactions don't necessarily need the energy from ATP-hydrolysis.<br /> <br /> The cleavage of DNA is achieved by a '''transesterification reaction between the tyrosines (Tyr122) and the target phosphoryl groups on opposing strands of the DNA backbone''', resulting in the tyrosine being covalentlty attached to the 5' end of the cleaved segment with a 4-base overhang.<br /> The '''active site tyrosines (Tyr 122)''' are on loops at either end of the dimer interface, 30Å apart, and sit at the ends of strongly basic grooves created by the dimer-related monomers.<br /> <br /> The gyrase structure reveals a '''new cluster of conserved residues''', juxtaposing Tyr 122 and Arg 121 from one monomer and His 80, Arg 32 and Lys 42 from the other monomer. '''This cluster may form the active site of the breakage–reunion reaction''', with the other conserved positive charges (Arg 46 and Arg 47) anchoring the non-covalently bound 3' end of the cleaved DNA.<br /> <br /> The gyrase could be inhibited by both the quinolones and the coumarins.<br /> Coumarins prevent the B-subunit of the gyrase from hydrolyzing the ATP, so that the gyrase can't come back to its starting state for another round of supercoiling. The mecanism is not yet well known.<br /> The quinolones inhibit DNA supercoiling by gyrase, it has even the ability to induce DNA cleavage instead. It appears that these drugs will arrest any gyrase rection involving double-strand DNA breakage.<br /> However, some mutations offers quinolone resistance, most of them involved a part of the GyrA sequence : between the amino acid 67 and 106. <br /> There are also others antibiotics, like the Cinodine which binds DNA and inhibits its supercoiling by gyrase.<br /> <br /> ==See Also==<br /> *[[Gyrase|Gyrase]]<br /> <br /> ==Reference==<br /> <ref group="xtra">PMID:009278055</ref><references group="xtra"/><br /> [[Category: Escherichia coli]]<br /> [[Category: Cabral, J H.M.]]<br /> [[Category: Liddington, R C.]]<br /> [[Category: Maxwell, A.]]<br /> [[Category: Gyrase]]<br /> [[Category: Supercoiling dna]]<br /> [[Category: Topoisomerase]]<br /> [[Category: Topoisomerase ii]]<br /> *Reece, Richard J., Anthony Maxwell, and James C. Wang. "DNA gyrase: structure and function." Critical reviews in biochemistry and molecular biology 26.3-4 (1991): 335-375.</div>

Laurent Thomas https://proteopedia.org/index.php?title=Sandbox_Reserved_828&diff=1881257 Sandbox Reserved 828 2013-12-29T22:12:33Z

<p>Laurent Thomas: </p> <hr /> <div><br /> {{Sandbox_Reserved_ESBS}}<br /> <br /> <br /> ----<br /> <Structure load='1ab4_mm1.pdb' size='400' frame='true' align='right' caption='Monomer of GyrA59 subunit at 2.8Å (1ab4.pdb)' scene='56/568026/Coloured/1'><br /> [[Image:1ab4.png|left|200px]]<br /> <br /> =='''Introduction :'''==<br /> <br /> <br /> Gyrase is the '''only prokaryote DNA topoisomerase II able to introduce negative supercoils in the DNA''' (see [http://en.wikipedia.org/wiki/DNA_supercoil DNA Supercoil]) in order to remove positive supercoils. It '''catalyses the hydrolysis of two phosphodiester bonds in a DNA segment (DNA cleavage)''' (called G segment). Then, thanks to '''ATP dependant conformation changes''' it enables the passage of another segment (the T segment) through the break, and then religates the broken segment. Gyrase acts prior to the replication (before the replication fork) or other mecanisms requiring loose DNA. <br /> '''In abscence of ATP, like other topoisomerases II, gyrase only relaxes supercoils.'''<br /> <br /> Gyrase is coded by two differents contiguous genes gyrA and GgyrB as it is a 350 kDa A<sub>2</sub>B<sub>2</sub> heterotetramers of two A proteins and two B proteins. <br /> The A protein breaks and religates DNA . The B protein has ATPase activity<br /> <br /> ----<br /> =='''Structure'''==<br /> <br /> [[Image:Gyrasegene.jpg]]<br /> <br /> '''The A protein''' breaks and religates DNA as the DNA cleavage core and the CTD lies on it. <br /> The gyrA 59kDa N-ter domain is called Breakage and reunion domain. It is composed of two domains at the <scene name='56/568026/Head/2'>head</scene> region :<br /> <br /> a winged-helix-turn-helix domain or <scene name='56/568026/Whd/1'>winged helix domain (WHD</scene>) where lies the catalytic tyrosines: the active-site tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> are on loops at either end of the dimer interface.<br /> <br /> a <scene name='56/568026/Tower/1'>tower domain</scene> with alpha/beta structure that participates in DNA bending during cleavage.<br /> <br /> The ‘head’ dimer interface is dominated by an antiparallel side by-side packing of the <scene name='56/568026/Alpha3/2'>alpha 3</scene> (the first helix of the HTH motif) from each monomer, together with their adjoining loops. At the top of the interface, the ‘recognition’ helices <scene name='56/568026/Alpha4/1'>alpha4</scene> make a head-to-head antiparallel dimer contact.<br /> <br /> <br /> There is a <scene name='56/568026/Coiledcoil_141819/1'>Coiled-coil</scene> domain, folowing the tower, with a helical core at the tail region. Two long helices (<br /> <scene name='56/568026/Helix14/1'>alpha helix 14</scene> and <scene name='56/568026/Helix18/1'>alpha helix 18</scene>) emanate from this core and connect, together with the C-terminal helix (<br /> <scene name='56/568026/Helix19/1'>alpha helix 19</scene>), the head and tail fragments. This domain has a small globular domain at it's end and is involved in the dimerization creating the <scene name='56/568026/Coiledcoil/1'>C-gate</scene> .<br /> <br /> <br /> The three connecting helices (α14,α18 and α19) adopt very different conformations, leading to large quaternary movements involving a single hinge-point within the helices and rigid body movements of <br /> the head fragments. <br /> <br /> [[Image:helixrot.jpg]] [[Image:gyrA592.jpg]]<br /> <br /> The tail is structurally conserved although large surface loops emanating from different points give it a different outward appearance. <br /> GyrA forms a heart-shaped homodimer with two protein interfaces, the DNA- and C-gates. GyrA59 is the minimal fragment of the A-subunit which, when complexed with the B-subunit, has DNA-cleavage activity. <br /> The remaining 30–35 kDa comprising the C-terminal domain (CTD) of GyrA shows a domain forming a b-pinwheel with a positively charged amino-acid perimeter. This carboxy-terminal domain of GyrA (cyan) is required for the introduction of DNA supercoils), DNA wraps around it. It is linked to the central part of Gyrase A by a flexible 13-15 residues region and stands close to the C-gate and may present an up and down movement in the early stades of the catalytic cycle and may be involved in the helping of G segment and D-gate binding so as the CTD binding of DNA.<br /> <br /> '''The B protein''' has the GHKL ATPase domain, a transducer domain and the Toprim fold on it. <br /> Two ATPase domains from two monomers dimerize to form a closed conformation, the N-gate. <br /> The Toprim fold is a Rossmann fold (beta-alpha-beta-alpha-beta) that contains three invariant acidic residues that coordinate magnesium ions involved in DNA cleavage and DNA religation. This torpim fold interacts with the Winged helix domain of gyrA to create the D-gate.<br /> <br /> <br /> '''The central core''' of the protein is made out of the association of GyrA and Gyr B with the Toprim fold, the DNA-binding domain (WHD)and tower domain, all three creating the D-gate.<br /> These domains are composed of numerous positively charged residues to interact with DNA.<br /> The Toprim domain is flexible and that this flexibility can allow the Toprim domain to coordinate with <br /> the WHD to form a competent cleavage complex.<br /> DNA is bent by ~150 degrees through an invariant isoleucine (I172)<br /> <br /> <br /> [[Image:Gyrasegates.jpg]]<br /> <br /> <br /> ----<br /> '''Mecanism'''<br /> <br /> 40-bp of duplex DNA, the G-segment, bind to the core of the enzyme and are cleaved by the active site tyrosines, while another DNA duplex, the T-segment, is captured through the ATP-induced dimerization of a protein gate, the N-gate. After passage through the transiently broken G-segment (DNA gate), the T-segment exits the protein through another protein gate, the C-gate. ATP hydrolysis and release reset the conformation of the enzyme and DNA to their initial state, poised for another strand-passage event or release of the DNA.<br /> N- and C-gates that open or close to allow T-segment transport through both theprotein and the cleaved G-segment.<br /> <br /> <br /> [[Image:mecanismgyrase.jpg]]<br /> <br /> ----<br /> <br /> =='''Reaction'''==<br /> <br /> The gyrase binds more strongly to relaxed or linear DNA than to supercoiled one by a factor of about ten.<br /> <br /> <br /> <br /> <br /> The cleavage of DNA is achieved by a '''transesterification reaction between the tyrosines (Tyr122) and the target phosphoryl groups on opposing strands of the DNA backbone''', resulting in the tyrosine being covalentlty attached to the 5' end of the cleaved segment with a 4-base overhang.<br /> The '''active site tyrosines (Tyr 122)''' are on loops at either end of the dimer interface, 30Å apart, and sit at the ends of strongly basic grooves created by the dimer-related monomers.<br /> <br /> The gyrase structure reveals a '''new cluster of conserved residues''', juxtaposing Tyr 122 and Arg 121 from one monomer and His 80, Arg 32 and Lys 42 from the other monomer. '''This cluster may form the active site of the breakage–reunion reaction''', with the other conserved positive charges (Arg 46 and Arg 47) anchoring the non-covalently bound 3' end of the cleaved DNA.<br /> <br /> The A subunit is responsible for the breakage reunion of DNA whereas the B-subunit has the ATPase activity.<br /> The introduction of negative supercoils requires the energy of ATP-hydrolysis. 2 ATP are hydrolyzed per reaction (2 B-subunits in the gyrase)so that the linking number (Lk) changes in step of 2. The ATPase activity of the B subunit is partially located in the N-terminal region of protein B whereas the C-terminal is involved in the interaction with the A subunit and DNA. <br /> The B subunit has a weak ATPase activity in the absence of A and DNA, eventhought the A subunit is able to bind DNA without the B subunit. Both subunits are required for all the reactions of gyrase.<br /> The DNA supercoiling reaction requires in addition to ATP, a divalent cation such as Mg<sup>2+</sup>, and is stimulated in the presence of spermidine. <br /> Therefore, the gyrase could also catalyse the following reaction :<br /> * Relaxation of negative/positive supercoils<br /> * Catenation/Decatenation of closed-circular DNA<br /> * DNA cleavage (induced by Quinolone)<br /> * Unknotting DNA strands<br /> Contrary to the introduction of negative supercoils, some of these reactions don't necessarily need the energy from ATP-hydrolysis.<br /> The gyrase could be inhibited by both the quinolones and the coumarins.<br /> Coumarins prevent the B-subunit of the gyrase from hydrolyzing the ATP, so that the gyrase can't come back to its starting state for another round of supercoiling. The mecanism is not yet well known.<br /> The quinolones inhibit DNA supercoiling by gyrase, it has even the ability to induce DNA cleavage instead. It appears that these drugs will arrest any gyrase rection involving double-strand DNA breakage.<br /> However, some mutations offers quinolone resistance, most of them involved a part of the GyrA sequence : between the amino acid 67 and 106. <br /> There are also others antibiotics, like the Cinodine which binds DNA and inhibits its supercoiling by gyrase.<br /> <br /> <br /> ==See Also==<br /> *[[Gyrase|Gyrase]]<br /> <br /> ==Reference==<br /> <ref group="xtra">PMID:009278055</ref><references group="xtra"/><br /> [[Category: Escherichia coli]]<br /> [[Category: Cabral, J H.M.]]<br /> [[Category: Liddington, R C.]]<br /> [[Category: Maxwell, A.]]<br /> [[Category: Gyrase]]<br /> [[Category: Supercoiling dna]]<br /> [[Category: Topoisomerase]]<br /> [[Category: Topoisomerase ii]]<br /> *Reece, Richard J., Anthony Maxwell, and James C. Wang. "DNA gyrase: structure and function." Critical reviews in biochemistry and molecular biology 26.3-4 (1991): 335-375.</div>

Laurent Thomas https://proteopedia.org/index.php?title=Sandbox_Reserved_828&diff=1881256 Sandbox Reserved 828 2013-12-29T20:12:57Z

<p>Laurent Thomas: </p> <hr /> <div><br /> {{Sandbox_Reserved_ESBS}}<br /> <br /> <br /> ----<br /> <Structure load='1ab4_mm1.pdb' size='400' frame='true' align='right' caption='Monomer of GyrA59 subunit at 2.8Å (1ab4.pdb)' scene='56/568026/Coloured/1'><br /> [[Image:1ab4.png|left|200px]]<br /> <br /> =='''Introduction :'''==<br /> <br /> <br /> Gyrase is the '''only prokaryote DNA topoisomerase II able to introduce negative supercoils in the DNA''' (see [http://en.wikipedia.org/wiki/DNA_supercoil DNA Supercoil]) in order to remove positive supercoils. It '''catalyses the hydrolysis of two phosphodiester bonds in a DNA segment''' (called G segment). Then, thanks to '''ATP dependant conformation changes''' it enables the passage of another segment (the T segment) through the break, and then religates the broken segment. Gyrase acts prior to the replication (before the replication fork) or other mecanisms requiring loose DNA. <br /> '''In abscence of ATP, like other topoisomerases II, gyrase only relaxes supercoils.'''<br /> <br /> Gyrase is coded by two differents contiguous genes gyrA and GgyrB as it is a 350 kDa A<sub>2</sub>B<sub>2</sub> heterotetramers of two A proteins and two B proteins. <br /> The A protein breaks and religates DNA . The B protein has ATPase activity<br /> <br /> ----<br /> =='''Structure'''==<br /> <br /> [[Image:Gyrasegene.jpg]]<br /> <br /> '''The A protein''' breaks and religates DNA as the DNA cleavage core and the CTD lies on it. <br /> The gyrA 59kDa N-ter domain is called Breakage and reunion domain. It is composed of two domains at the <scene name='56/568026/Head/2'>head</scene> region :<br /> <br /> a winged-helix-turn-helix domain or <scene name='56/568026/Whd/1'>winged helix domain (WHD</scene>) where lies the catalytic tyrosines: the active-site tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> are on loops at either end of the dimer interface.<br /> <br /> a <scene name='56/568026/Tower/1'>tower domain</scene> with alpha/beta structure that participates in DNA bending during cleavage.<br /> <br /> The ‘head’ dimer interface is dominated by an antiparallel side by-side packing of the <scene name='56/568026/Alpha3/2'>alpha 3</scene> (the first helix of the HTH motif) from each monomer, together with their adjoining loops. At the top of the interface, the ‘recognition’ helices <scene name='56/568026/Alpha4/1'>alpha4</scene> make a head-to-head antiparallel dimer contact.<br /> <br /> <br /> There is a <scene name='56/568026/Coiledcoil_141819/1'>Coiled-coil</scene> domain, folowing the tower, with a helical core at the tail region. Two long helices (<br /> <scene name='56/568026/Helix14/1'>alpha helix 14</scene> and <scene name='56/568026/Helix18/1'>alpha helix 18</scene>) emanate from this core and connect, together with the C-terminal helix (<br /> <scene name='56/568026/Helix19/1'>alpha helix 19</scene>), the head and tail fragments. This domain has a small globular domain at it's end and is involved in the dimerization creating the <scene name='56/568026/Coiledcoil/1'>C-gate</scene> .<br /> <br /> <br /> The three connecting helices (α14,α18 and α19) adopt very different conformations, leading to large quaternary movements involving a single hinge-point within the helices and rigid body movements of <br /> the head fragments. <br /> <br /> [[Image:helixrot.jpg]] [[Image:gyrA592.jpg]]<br /> <br /> The tail is structurally conserved although large surface loops emanating from different points give it a different outward appearance. <br /> GyrA forms a heart-shaped homodimer with two protein interfaces, the DNA- and C-gates. GyrA59 is the minimal fragment of the A-subunit which, when complexed with the B-subunit, has DNA-cleavage activity. <br /> The remaining 30–35 kDa comprising the C-terminal domain (CTD) of GyrA shows a domain forming a b-pinwheel with a positively charged amino-acid perimeter. This carboxy-terminal domain of GyrA (cyan) is required for the introduction of DNA supercoils), DNA wraps around it. It is linked to the central part of Gyrase A by a flexible 13-15 residues region and stands close to the C-gate and may present an up and down movement in the early stades of the catalytic cycle and may be involved in the helping of G segment and D-gate binding so as the CTD binding of DNA.<br /> <br /> '''The B protein''' has the GHKL ATPase domain, a transducer domain and the Toprim fold on it. <br /> Two ATPase domains from two monomers dimerize to form a closed conformation, the N-gate. <br /> The Toprim fold is a Rossmann fold (beta-alpha-beta-alpha-beta) that contains three invariant acidic residues that coordinate magnesium ions involved in DNA cleavage and DNA religation. This torpim fold interacts with the Winged helix domain of gyrA to create the D-gate.<br /> <br /> <br /> '''The central core''' of the protein is made out of the association of GyrA and Gyr B with the Toprim fold, the DNA-binding domain (WHD)and tower domain, all three creating the D-gate.<br /> These domains are composed of numerous positively charged residues to interact with DNA.<br /> The Toprim domain is flexible and that this flexibility can allow the Toprim domain to coordinate with <br /> the WHD to form a competent cleavage complex.<br /> DNA is bent by ~150 degrees through an invariant isoleucine (I172)<br /> <br /> <br /> [[Image:Gyrasegates.jpg]]<br /> <br /> <br /> ----<br /> '''Mecanism'''<br /> <br /> 40-bp of duplex DNA, the G-segment, bind to the core of the enzyme and are cleaved by the active site tyrosines, while another DNA duplex, the T-segment, is captured through the ATP-induced dimerization of a protein gate, the N-gate. After passage through the transiently broken G-segment (DNA gate), the T-segment exits the protein through another protein gate, the C-gate. ATP hydrolysis and release reset the conformation of the enzyme and DNA to their initial state, poised for another strand-passage event or release of the DNA.<br /> N- and C-gates that open or close to allow T-segment transport through both theprotein and the cleaved G-segment.<br /> <br /> <br /> [[Image:mecanismgyrase.jpg]]<br /> <br /> ----<br /> <br /> =='''Reaction'''==<br /> The cleavage of DNA is achieved by a '''transesterification reaction between the tyrosines (Tyr122) and the target phosphoryl groups on opposing strands of the DNA backbone''', resulting in the tyrosine being covalentlty attached to the 5' end of the cleaved segment with a 4-base overhang.<br /> The '''active site tyrosines (Tyr 122)''' are on loops at either end of the dimer interface, 30Å apart, and sit at the ends of strongly basic grooves created by the dimer-related monomers.<br /> <br /> The gyrase structure reveals a '''new cluster of conserved residues''', juxtaposing Tyr 122 and Arg 121 from one monomer and His 80, Arg 32 and Lys 42 from the other monomer. '''This cluster may form the active site of the breakage–reunion reaction''', with the other conserved positive charges (Arg 46 and Arg 47) anchoring the non-covalently bound 3' end of the cleaved DNA.<br /> <br /> The main reaction catalysed by the gyrase is the DNa cleavage. It requires the energy of ATP-Hydrolisis. 2 ATP are hydrolised per reaction (2 B-subunits in the gyrase)so that the linking number (Lk) changes in step of 2.<br /> The DNA supercoiling reaction requires in addition to ATP, a divalent cation such as Mg<sup>2+</sup>, and is stimulated in the presence of spermidine. <br /> Therefore, the gyrase could also catalyse the following reaction :<br /> *<br /> <br /> <br /> ==See Also==<br /> *[[Gyrase|Gyrase]]<br /> <br /> ==Reference==<br /> <ref group="xtra">PMID:009278055</ref><references group="xtra"/><br /> [[Category: Escherichia coli]]<br /> [[Category: Cabral, J H.M.]]<br /> [[Category: Liddington, R C.]]<br /> [[Category: Maxwell, A.]]<br /> [[Category: Gyrase]]<br /> [[Category: Supercoiling dna]]<br /> [[Category: Topoisomerase]]<br /> [[Category: Topoisomerase ii]]<br /> *Reece, Richard J., Anthony Maxwell, and James C. Wang. "DNA gyrase: structure and function." Critical reviews in biochemistry and molecular biology 26.3-4 (1991): 335-375.</div>

Laurent Thomas https://proteopedia.org/index.php?title=Sandbox_Reserved_828&diff=1881255 Sandbox Reserved 828 2013-12-29T20:12:19Z

<p>Laurent Thomas: </p> <hr /> <div><br /> {{Sandbox_Reserved_ESBS}}<br /> <br /> <br /> ----<br /> <Structure load='1ab4_mm1.pdb' size='400' frame='true' align='right' caption='Monomer of GyrA59 subunit at 2.8Å (1ab4.pdb)' scene='56/568026/Coloured/1'><br /> [[Image:1ab4.png|left|200px]]<br /> <br /> =='''Introduction :'''==<br /> <br /> <br /> Gyrase is the '''only prokaryote DNA topoisomerase II able to introduce negative supercoils in the DNA''' (see [http://en.wikipedia.org/wiki/DNA_supercoil DNA Supercoil]) in order to remove positive supercoils. It '''catalyses the hydrolysis of two phosphodiester bonds in a DNA segment''' (called G segment). Then, thanks to '''ATP dependant conformation changes''' it enables the passage of another segment (the T segment) through the break, and then religates the broken segment. Gyrase acts prior to the replication (before the replication fork) or other mecanisms requiring loose DNA. <br /> '''In abscence of ATP, like other topoisomerases II, gyrase only relaxes supercoils.'''<br /> <br /> Gyrase is coded by two differents contiguous genes gyrA and GgyrB as it is a 350 kDa A<sub>2</sub>B<sub>2</sub> heterotetramers of two A proteins and two B proteins. <br /> The A protein breaks and religates DNA . The B protein has ATPase activity<br /> <br /> ----<br /> =='''Structure'''==<br /> <br /> [[Image:Gyrasegene.jpg]]<br /> <br /> '''The A protein''' breaks and religates DNA as the DNA cleavage core and the CTD lies on it. <br /> The gyrA 59kDa N-ter domain is called Breakage and reunion domain. It is composed of two domains at the <scene name='56/568026/Head/2'>head</scene> region :<br /> <br /> a winged-helix-turn-helix domain or <scene name='56/568026/Whd/1'>winged helix domain (WHD</scene>) where lies the catalytic tyrosines: the active-site tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> are on loops at either end of the dimer interface.<br /> <br /> a <scene name='56/568026/Tower/1'>tower domain</scene> with alpha/beta structure that participates in DNA bending during cleavage.<br /> <br /> The ‘head’ dimer interface is dominated by an antiparallel side by-side packing of the <scene name='56/568026/Alpha3/2'>alpha 3</scene> (the first helix of the HTH motif) from each monomer, together with their adjoining loops. At the top of the interface, the ‘recognition’ helices <scene name='56/568026/Alpha4/1'>alpha4</scene> make a head-to-head antiparallel dimer contact.<br /> <br /> <br /> There is a <scene name='56/568026/Coiledcoil_141819/1'>Coiled-coil</scene> domain, folowing the tower, with a helical core at the tail region. Two long helices (<br /> <scene name='56/568026/Helix14/1'>alpha helix 14</scene> and <scene name='56/568026/Helix18/1'>alpha helix 18</scene>) emanate from this core and connect, together with the C-terminal helix (<br /> <scene name='56/568026/Helix19/1'>alpha helix 19</scene>), the head and tail fragments. This domain has a small globular domain at it's end and is involved in the dimerization creating the <scene name='56/568026/Coiledcoil/1'>C-gate</scene> .<br /> <br /> <br /> The three connecting helices (α14,α18 and α19) adopt very different conformations, leading to large quaternary movements involving a single hinge-point within the helices and rigid body movements of <br /> the head fragments. <br /> <br /> [[Image:helixrot.jpg]] [[Image:gyrA592.jpg]]<br /> <br /> The tail is structurally conserved although large surface loops emanating from different points give it a different outward appearance. <br /> GyrA forms a heart-shaped homodimer with two protein interfaces, the DNA- and C-gates. GyrA59 is the minimal fragment of the A-subunit which, when complexed with the B-subunit, has DNA-cleavage activity. <br /> The remaining 30–35 kDa comprising the C-terminal domain (CTD) of GyrA shows a domain forming a b-pinwheel with a positively charged amino-acid perimeter. This carboxy-terminal domain of GyrA (cyan) is required for the introduction of DNA supercoils), DNA wraps around it. It is linked to the central part of Gyrase A by a flexible 13-15 residues region and stands close to the C-gate and may present an up and down movement in the early stades of the catalytic cycle and may be involved in the helping of G segment and D-gate binding so as the CTD binding of DNA.<br /> <br /> '''The B protein''' has the GHKL ATPase domain, a transducer domain and the Toprim fold on it. <br /> Two ATPase domains from two monomers dimerize to form a closed conformation, the N-gate. <br /> The Toprim fold is a Rossmann fold (beta-alpha-beta-alpha-beta) that contains three invariant acidic residues that coordinate magnesium ions involved in DNA cleavage and DNA religation. This torpim fold interacts with the Winged helix domain of gyrA to create the D-gate.<br /> <br /> <br /> '''The central core''' of the protein is made out of the association of GyrA and Gyr B with the Toprim fold, the DNA-binding domain (WHD)and tower domain, all three creating the D-gate.<br /> These domains are composed of numerous positively charged residues to interact with DNA.<br /> The Toprim domain is flexible and that this flexibility can allow the Toprim domain to coordinate with <br /> the WHD to form a competent cleavage complex.<br /> DNA is bent by ~150 degrees through an invariant isoleucine (I172)<br /> <br /> <br /> [[Image:Gyrasegates.jpg]]<br /> <br /> <br /> ----<br /> '''Mecanism'''<br /> <br /> 40-bp of duplex DNA, the G-segment, bind to the core of the enzyme and are cleaved by the active site tyrosines, while another DNA duplex, the T-segment, is captured through the ATP-induced dimerization of a protein gate, the N-gate. After passage through the transiently broken G-segment (DNA gate), the T-segment exits the protein through another protein gate, the C-gate. ATP hydrolysis and release reset the conformation of the enzyme and DNA to their initial state, poised for another strand-passage event or release of the DNA.<br /> N- and C-gates that open or close to allow T-segment transport through both theprotein and the cleaved G-segment.<br /> <br /> <br /> [[Image:mecanismgyrase.jpg]]<br /> <br /> ----<br /> <br /> =='''Reaction'''==<br /> The cleavage of DNA is achieved by a '''transesterification reaction between the tyrosines (Tyr122) and the target phosphoryl groups on opposing strands of the DNA backbone''', resulting in the tyrosine being covalentlty attached to the 5' end of the cleaved segment with a 4-base overhang.<br /> The '''active site tyrosines (Tyr 122)''' are on loops at either end of the dimer interface, 30Å apart, and sit at the ends of strongly basic grooves created by the dimer-related monomers.<br /> <br /> The gyrase structure reveals a '''new cluster of conserved residues''', juxtaposing Tyr 122 and Arg 121 from one monomer and His 80, Arg 32 and Lys 42 from the other monomer. '''This cluster may form the active site of the breakage–reunion reaction''', with the other conserved positive charges (Arg 46 and Arg 47) anchoring the non-covalently bound 3' end of the cleaved DNA.<br /> <br /> The main reaction catalysed by the gyrase is the DNa cleavage. It requires the energy of ATP-Hydrolisis. 2 ATP are hydrolised per reaction (2 B-subunits in the gyrase)so that the linking number (Lk) changes in step of 2.<br /> The DNA supercoiling reaction requires in addition to ATP, a divalent cation such as Mg<sup>2+</sup>, and is stimulated in the presence of spermidine. <br /> Therefore, the gyrase could also catalyse the following reaction :<br /> *<br /> <br /> <br /> ==See Also==<br /> *[[Gyrase|Gyrase]]<br /> <br /> ==Reference==<br /> <ref group="xtra">PMID:009278055</ref><references group="xtra"/><br /> [[Category: Escherichia coli]]<br /> [[Category: Cabral, J H.M.]]<br /> [[Category: Liddington, R C.]]<br /> [[Category: Maxwell, A.]]<br /> [[Category: Gyrase]]<br /> [[Category: Supercoiling dna]]<br /> [[Category: Topoisomerase]]<br /> [[Category: Topoisomerase ii]]<br /> •Reece, Richard J., Anthony Maxwell, and James C. Wang. "DNA gyrase: structure and function." Critical reviews in biochemistry and molecular biology 26.3-4 (1991): 335-375.</div>

Laurent Thomas https://proteopedia.org/index.php?title=Sandbox_Reserved_828&diff=1881252 Sandbox Reserved 828 2013-12-29T19:47:00Z

<p>Laurent Thomas: </p> <hr /> <div><br /> {{Sandbox_Reserved_ESBS}}<br /> <br /> <br /> ----<br /> <Structure load='1ab4_mm1.pdb' size='400' frame='true' align='right' caption='Monomer of GyrA59 subunit at 2.8Å (1ab4.pdb)' scene='56/568026/Coloured/1'><br /> [[Image:1ab4.png|left|200px]]<br /> <br /> =='''Introduction :'''==<br /> <br /> <br /> Gyrase is the '''only prokaryote DNA topoisomerase II able to introduce negative supercoils in the DNA''' in order to remove positive supercoils. It '''catalyses the hydrolysis of two phosphodiester bonds in a DNA segment''' (called G segment). Then, thanks to '''ATP dependant conformation changes''' it enables the passage of another segment (the T segment) through the break, and then religates the broken segment. Gyrase acts prior to the replication (before the replication fork) or other mecanisms requiring loose DNA. <br /> '''In abscence of ATP, like other topoisomerases II, gyrase only relaxes supercoils.'''<br /> <br /> Gyrase is coded by two differents contiguous genes gyrA and GgyrB as it is a 350 kDa A2B2 heterotetramers of two A proteins and two B proteins. <br /> The A protein breaks and religates DNA . The B protein has ATPase activity<br /> <br /> ----<br /> =='''Structure'''==<br /> <br /> [[Image:Gyrasegene.jpg]]<br /> <br /> '''The A protein''' breaks and religates DNA as the DNA cleavage core and the CTD lies on it. <br /> The gyrA 59kDa N-ter domain is called Breakage and reunion domain. It is composed of two domains at the <scene name='56/568026/Head/2'>head</scene> region :<br /> <br /> a winged-helix-turn-helix domain or <scene name='56/568026/Whd/1'>winged helix domain (WHD</scene>) where lies the catalytic tyrosines: the active-site tyrosines <scene name='56/568026/Tyr122/1'>(Tyr 122)</scene> are on loops at either end of the dimer interface.<br /> <br /> a <scene name='56/568026/Tower/1'>tower domain</scene> with alpha/beta structure that participates in DNA bending during cleavage.<br /> <br /> The ‘head’ dimer interface is dominated by an antiparallel side by-side packing of the <scene name='56/568026/Alpha3/2'>alpha 3</scene> (the first helix of the HTH motif) from each monomer, together with their adjoining loops. At the top of the interface, the ‘recognition’ helices <scene name='56/568026/Alpha4/1'>alpha4</scene> make a head-to-head antiparallel dimer contact.<br /> <br /> <br /> There is a <scene name='56/568026/Coiledcoil_141819/1'>Coiled-coil</scene> domain, folowing the tower, with a helical core at the tail region. Two long helices (<br /> <scene name='56/568026/Helix14/1'>alpha helix 14</scene> and <scene name='56/568026/Helix18/1'>alpha helix 18</scene>) emanate from this core and connect, together with the C-terminal helix (<br /> <scene name='56/568026/Helix19/1'>alpha helix 19</scene>), the head and tail fragments. This domain has a small globular domain at it's end and is involved in the dimerization creating the <scene name='56/568026/Coiledcoil/1'>C-gate</scene> .<br /> <br /> <br /> The three connecting helices (a14,a18 anda19) adopt very different conformations, leading to large quaternary movements involving a single hinge-point within the helices and rigid body movements of <br /> the head fragments. <br /> <br /> [[Image:helixrot.jpg]] [[Image:gyrA592.jpg]]<br /> <br /> The tail is structurally conserved although large surface loops emanating from different points give it a different outward appearance. <br /> GyrA forms a heart-shaped homodimer with two protein interfaces, the DNA- and C-gates. GyrA59 is the minimal fragment of the A-subunit which, when complexed with the B-subunit, has DNA-cleavage activity. <br /> The remaining 30–35 kDa comprising the C-terminal domain (CTD) of GyrA shows a domain forming a b-pinwheel with a positively charged amino-acid perimeter. This carboxy-terminal domain of GyrA (cyan) is required for the introduction of DNA supercoils), DNA wraps around it. It is linked to the central part of Gyrase A by a flexible 13-15 residues region and stands close to the C-gate and may present an up and down movement in the early stades of the catalytic cycle and may be involved in the helping of G segment and D-gate binding so as the CTD binding of DNA.<br /> <br /> '''The B protein''' has the GHKL ATPase domain, a transducer domain and the Toprim fold on it. <br /> Two ATPase domains from two monomers dimerize to form a closed conformation, the N-gate. <br /> The Toprim fold is a Rossmann fold (beta-alpha-beta-alpha-beta) that contains three invariant acidic residues that coordinate magnesium ions involved in DNA cleavage and DNA religation. This torpim fold interacts with the Winged helix domain of gyrA to create the D-gate.<br /> <br /> <br /> '''The central core''' of the protein is made out of the association of GyrA and Gyr B with the Toprim fold, the DNA-binding domain (WHD)and tower domain, all three creating the D-gate.<br /> These domains are composed of numerous positively charged residues to interact with DNA.<br /> The Toprim domain is flexible and that this flexibility can allow the Toprim domain to coordinate with <br /> the WHD to form a competent cleavage complex.<br /> DNA is bent by ~150 degrees through an invariant isoleucine (I172)<br /> <br /> <br /> [[Image:Gyrasegates.jpg]]<br /> <br /> <br /> ----<br /> '''Mecanism'''<br /> <br /> 40-bp of duplex DNA, the G-segment, bind to the core of the enzyme and are cleaved by the active site tyrosines, while another DNA duplex, the T-segment, is captured through the ATP-induced dimerization of a protein gate, the N-gate. After passage through the transiently broken G-segment (DNA gate), the T-segment exits the protein through another protein gate, the C-gate. ATP hydrolysis and release reset the conformation of the enzyme and DNA to their initial state, poised for another strand-passage event or release of the DNA.<br /> N- and C-gates that open or close to allow T-segment transport through both theprotein and the cleaved G-segment.<br /> <br /> <br /> [[Image:mecanismgyrase.jpg]]<br /> <br /> ----<br /> <br /> =='''Reaction'''==<br /> The cleavage of DNA is achieved by a '''transesterification reaction between the tyrosines (Tyr122) and the target phosphoryl groups on opposing strands of the DNA backbone''', resulting in the tyrosine being covalentlty attached to the 5' end of the cleaved segment with a 4-base overhang.<br /> The '''active site tyrosines (Tyr 122)''' are on loops at either end of the dimer interface, 30Å apart, and sit at the ends of strongly basic grooves created by the dimer-related monomers.<br /> <br /> The gyrase structure reveals a '''new cluster of conserved residues''', juxtaposing Tyr 122 and Arg 121 from one monomer and His 80, Arg 32 and Lys 42 from the other monomer. '''This cluster may form the active site of the breakage–reunion reaction''', with the other conserved positive charges (Arg 46 and Arg 47) anchoring the non-covalently bound 3' end of the cleaved DNA.<br /> <br /> ==See Also==<br /> *[[Gyrase|Gyrase]]<br /> <br /> ==Reference==<br /> <ref group="xtra">PMID:009278055</ref><references group="xtra"/><br /> [[Category: Escherichia coli]]<br /> [[Category: Cabral, J H.M.]]<br /> [[Category: Liddington, R C.]]<br /> [[Category: Maxwell, A.]]<br /> [[Category: Gyrase]]<br /> [[Category: Supercoiling dna]]<br /> [[Category: Topoisomerase]]<br /> [[Category: Topoisomerase ii]]<br /> •Reece, Richard J., Anthony Maxwell, and James C. Wang. "DNA gyrase: structure and function." Critical reviews in biochemistry and molecular biology 26.3-4 (1991): 335-375.</div>

Laurent Thomas https://proteopedia.org/index.php?title=Sandbox_Reserved_828&diff=1878950 Sandbox Reserved 828 2013-12-24T16:17:13Z

<p>Laurent Thomas: </p> <hr /> <div><br /> {{Sandbox_Reserved_ESBS}}<br /> <br /> <br /> ----<br /> <Structure load='1ab4' size='400' frame='true' align='right' caption='Monomer of GyrA59 subunit at 2.8Å (1ab4.pdb)' scene='56/568026/Coloured/1'><br /> [[Image:1ab4.png|left|200px]]<br /> <br /> =='''Introduction :'''==<br /> <br /> <br /> Gyrase is the '''only prokaryote DNA topoisomerase II able to introduce negative supercoils in the DNA''' in order to remove positive supercoils. It '''catalyses the hydrolysis of two phosphodiester bonds in a DNA segment''' (called G segment). Then, thanks to '''ATP dependant conformation changes''' it enables the passage of another segment (the T segment) through the break, and then religates the broken segment. Gyrase acts prior to the replication (before the replication fork) or other mecanisms requiring loose DNA. <br /> '''In abscence of ATP, like other topoisomerases II, gyrase only relaxes supercoils.'''<br /> <br /> Gyrase is coded by two differents contiguous genes gyrA and GgyrB as it is a 350 kDa A2B2 heterotetramers of two A proteins and two B proteins. <br /> The A protein breaks and religates DNA . The B protein has ATPase activity<br /> <br /> ----<br /> =='''Structure'''==<br /> <br /> [[Image:Gyrasegene.jpg]]<br /> <br /> '''The A protein''' breaks and religates DNA as the DNA cleavage core and the CTD lies on it. <br /> The gyrA 59kDa N-ter domain is called Breakage and reunion domain It is composed of two domains at the head region : a winged-helix-turn-helix domain or winged helix domain (WHD) where lies the catalytic tyrosines and a tower domain with alpha/beta structure.<br /> And a single domain with a helical core at the tail region. Two long helices (a14 anda18) emanate from this core and connect, together with the C-terminal helix (a19), the head and tail fragments.<br /> <br /> <br /> [[Image:gyrA59.jpg]]<br /> <br /> <br /> The three connecting helices (a14,a18 anda19) adopt very different conformations, leading to large quaternary movements involving a single hinge-point within the helices and rigid body movements of <br /> the head fragments. <br /> <br /> [[Image:helixrot.jpg]] [[Image:gyrA592.jpg]]<br /> <br /> The tail is structurally conserved although large surface loops emanating from different points give it a different outward appearance It forms a heart-shaped homodimer with two protein interfaces, the DNA- and C-gates. GyrA59 is the minimal fragment of the A-subunit which, when complexed with the B-subunit, has DNA-cleavage activity. <br /> The remaining 30–35 kDa comprising the C-terminal domain (CTD) of GyrA shows a domain forming a b-pinwheel with a positively charged amino-acid perimeter. This carboxy-terminal domain of GyrA (cyan) is required for the introduction of DNA supercoils). <br /> <br /> <br /> The B protein has the ATPase domain and the Toprim fold on it. Two ATPase domains dimerize to form a closed conformation. The Toprim fold is a Rossmann fold that contains three invariant acidic residues that coordinate magnesium ions involved in DNA cleavage and DNA religation<br /> The central core of the protein contains a Toprim fold and a DNA-binding core that contains a winged helix domain (WHD), often referred to as a CAP domain. The catalytic tyrosine lies on this WHD. The DNA-binding core consists of the WHD, which leads to a tower domain. . A coiled-coil region leads to a C-terminal domain that forms the main dimer interface<br /> the Toprim domain is flexible and that this flexibility can allow the Toprim domain to coordinate with the WHD to form a competent cleavage complex<br /> DNA was bent by ~150 degrees through an invariant isoleucine (in topoisomerase II it is I833 and in gyrase it is I172)<br /> The first structure of a C-terminal domain of gyrase was solved by Corbett et al. (Proceedings of the National Academy of Science, 2004, PDB ID = 1SUU)<br /> The structures formed a novel beta barrel, which bends DNA by wrapping the nucleic acid around itself. The bending of DNA by gyrase has been proposed as a key mechanism in the ability of gyrase to introduce negative supercoils into the DNA. This is consistent with footprinting data that shows <br /> that gyrase has a 140-base-pair footprint. Both gyrase and topoisomerase IV CTDs bend DNA, but only gyrase introduces negative supercoils.<br /> <br /> ----<br /> <br /> =='''Reaction'''==<br /> The cleavage of DNA is achieved by a '''transesterification reaction between the tyrosines (Tyr122) and the target phosphoryl groups on opposing strands of the DNA backbone''', resulting in the tyrosine being covalentlty attached to the 5' end of the cleaved segment with a 4-base overhang.<br /> The '''active site tyrosines (Tyr 122)''' are on loops at either end of the dimer interface, 30Å apart, and sit at the ends of strongly basic grooves created by the dimer-related monomers.<br /> <br /> The gyrase structure reveals a '''new cluster of conserved residues''', juxtaposing Tyr 122 and Arg 121 from one monomer and His 80, Arg 32 and Lys 42 from the other monomer. '''This cluster may form the active site of the breakage–reunion reaction''', with the other conserved positive charges (Arg 46 and Arg 47) anchoring the non-covalently bound 3' end of the cleaved DNA.<br /> <Structure load='1ab4_mm1.pdb' size='300' frame='true' align='right' caption='GyrA59, Breakage/Reunion subunit (dimer)' scene='56/568026/Biounit/1' /></div>

Laurent Thomas https://proteopedia.org/index.php?title=Sandbox_Reserved_828&diff=1878946 Sandbox Reserved 828 2013-12-24T16:12:23Z

<p>Laurent Thomas: </p> <hr /> <div><br /> {{Sandbox_Reserved_ESBS}}<br /> <br /> <br /> ----<br /> <Structure load='1ab4' size='400' frame='true' align='right' caption='Breakage/Reunion domain, monomer of A subunit at 2.8Å (1ab4.pdb)' scene='56/568026/Coloured/1'><br /> [[Image:1ab4.png|left|200px]]<br /> <br /> =='''Introduction :'''==<br /> <br /> <br /> Gyrase is the '''only prokaryote DNA topoisomerase II able to introduce negative supercoils in the DNA''' in order to remove positive supercoils. It '''catalyses the hydrolysis of two phosphodiester bonds in a DNA segment''' (called G segment). Then, thanks to '''ATP dependant conformation changes''' it enables the passage of another segment (the T segment) through the break, and then religates the broken segment. Gyrase acts prior to the replication (before the replication fork) or other mecanisms requiring loose DNA. <br /> '''In abscence of ATP, like other topoisomerases II, gyrase only relaxes supercoils.'''<br /> <br /> Gyrase is coded by two differents contiguous genes gyrA and GgyrB as it is a 350 kDa A2B2 heterotetramers of two A proteins and two B proteins. <br /> The A protein breaks and religates DNA . The B protein has ATPase activity<br /> <br /> ----<br /> =='''Structure'''==<br /> <br /> [[Image:Gyrasegene.jpg]]<br /> <br /> '''The A protein''' breaks and religates DNA as the DNA cleavage core and the CTD lies on it. <br /> The gyrA 59kDa N-ter domain is called Breakage and reunion domain It is composed of two domains at the head region : a winged-helix-turn-helix domain or winged helix domain (WHD) where lies the catalytic tyrosines and a tower domain with alpha/beta structure.<br /> And a single domain with a helical core at the tail region. Two long helices (a14 anda18) emanate from this core and connect, together with the C-terminal helix (a19), the head and tail fragments. <br /> The three connecting helices (a14,a18 anda19) adopt very different conformations, leading to large quaternary movements involving a single hinge-point within the helices and rigid body movements of <br /> the head fragments. <br /> [[Image:gyrA59.jpg]]<br /> The tail is structurally conserved although large surface loops emanating from different points give it a different outward appearance It forms a heart-shaped homodimer with two protein interfaces, the DNA- and C-gates. GyrA59 is the minimal fragment of the A-subunit which, when complexed with the B-subunit, has DNA-cleavage activity. <br /> The remaining 30–35 kDa comprising the C-terminal domain (CTD) of GyrA shows a domain forming a b-pinwheel with a positively charged amino-acid perimeter. This carboxy-terminal domain of GyrA (cyan) is required for the introduction of DNA supercoils). <br /> <br /> <br /> The B protein has the ATPase domain and the Toprim fold on it. Two ATPase domains dimerize to form a closed conformation. The Toprim fold is a Rossmann fold that contains three invariant acidic residues that coordinate magnesium ions involved in DNA cleavage and DNA religation<br /> The central core of the protein contains a Toprim fold and a DNA-binding core that contains a winged helix domain (WHD), often referred to as a CAP domain. The catalytic tyrosine lies on this WHD. The DNA-binding core consists of the WHD, which leads to a tower domain. . A coiled-coil region leads to a C-terminal domain that forms the main dimer interface<br /> the Toprim domain is flexible and that this flexibility can allow the Toprim domain to coordinate with the WHD to form a competent cleavage complex<br /> DNA was bent by ~150 degrees through an invariant isoleucine (in topoisomerase II it is I833 and in gyrase it is I172)<br /> The first structure of a C-terminal domain of gyrase was solved by Corbett et al. (Proceedings of the National Academy of Science, 2004, PDB ID = 1SUU)<br /> The structures formed a novel beta barrel, which bends DNA by wrapping the nucleic acid around itself. The bending of DNA by gyrase has been proposed as a key mechanism in the ability of gyrase to introduce negative supercoils into the DNA. This is consistent with footprinting data that shows <br /> that gyrase has a 140-base-pair footprint. Both gyrase and topoisomerase IV CTDs bend DNA, but only gyrase introduces negative supercoils.<br /> <br /> ----<br /> <br /> =='''Reaction'''==<br /> The cleavage of DNA is achieved by a '''transesterification reaction between the tyrosines (Tyr122) and the target phosphoryl groups on opposing strands of the DNA backbone''', resulting in the tyrosine being covalentlty attached to the 5' end of the cleaved segment with a 4-base overhang.<br /> The '''active site tyrosines (Tyr 122)''' are on loops at either end of the dimer interface, 30Å apart, and sit at the ends of strongly basic grooves created by the dimer-related monomers.<br /> <br /> The gyrase structure reveals a '''new cluster of conserved residues''', juxtaposing Tyr 122 and Arg 121 from one monomer and His 80, Arg 32 and Lys 42 from the other monomer. '''This cluster may form the active site of the breakage–reunion reaction''', with the other conserved positive charges (Arg 46 and Arg 47) anchoring the non-covalently bound 3' end of the cleaved DNA.<br /> <Structure load='1ab4_mm1.pdb' size='300' frame='true' align='right' caption='A subunit(dimer)' scene='56/568026/Biounit/1' /></div>

Laurent Thomas https://proteopedia.org/index.php?title=Sandbox_Reserved_828&diff=1878944 Sandbox Reserved 828 2013-12-24T16:10:20Z

<p>Laurent Thomas: </p> <hr /> <div><br /> {{Sandbox_Reserved_ESBS}}<br /> <br /> <br /> ----<br /> <Structure load='1ab4' size='400' frame='true' align='right' caption='Breakage/Reunion domain at 2.8Å (1ab4.pdb)' scene='56/568026/Coloured/1'><br /> [[Image:1ab4.png|left|200px]]<br /> <br /> =='''Introduction :'''==<br /> <br /> <br /> Gyrase is the '''only prokaryote DNA topoisomerase II able to introduce negative supercoils in the DNA''' in order to remove positive supercoils. It '''catalyses the hydrolysis of two phosphodiester bonds in a DNA segment''' (called G segment). Then, thanks to '''ATP dependant conformation changes''' it enables the passage of another segment (the T segment) through the break, and then religates the broken segment. Gyrase acts prior to the replication (before the replication fork) or other mecanisms requiring loose DNA. <br /> '''In abscence of ATP, like other topoisomerases II, gyrase only relaxes supercoils.'''<br /> <br /> Gyrase is coded by two differents contiguous genes gyrA and GgyrB as it is a 350 kDa A2B2 heterotetramers of two A proteins and two B proteins. <br /> The A protein breaks and religates DNA . The B protein has ATPase activity<br /> <br /> ----<br /> =='''Structure'''==<br /> <br /> [[Image:Gyrasegene.jpg]]<br /> <br /> '''The A protein''' breaks and religates DNA as the DNA cleavage core and the CTD lies on it. <br /> The gyrA 59kDa N-ter domain is called Breakage and reunion domain It is composed of two domains at the head region : a winged-helix-turn-helix domain or winged helix domain (WHD) where lies the catalytic tyrosines and a tower domain with alpha/beta structure.<br /> And a single domain with a helical core at the tail region. Two long helices (a14 anda18) emanate from this core and connect, together with the C-terminal helix (a19), the head and tail fragments. <br /> The three connecting helices (a14,a18 anda19) adopt very different conformations, leading to large quaternary movements involving a single hinge-point within the helices and rigid body movements of <br /> the head fragments . <br /> The tail is structurally conserved although large surface loops emanating from different points give it a different outward appearance It forms a heart-shaped homodimer with two protein interfaces, the DNA- and C-gates. GyrA59 is the minimal fragment of the A-subunit which, when complexed with the B-subunit, has DNA-cleavage activity. <br /> The remaining 30–35 kDa comprising the C-terminal domain (CTD) of GyrA shows a domain forming a b-pinwheel with a positively charged amino-acid perimeter. This carboxy-terminal domain of GyrA (cyan) is required for the introduction of DNA supercoils). <br /> <br /> <br /> The B protein has the ATPase domain and the Toprim fold on it. Two ATPase domains dimerize to form a closed conformation. The Toprim fold is a Rossmann fold that contains three invariant acidic residues that coordinate magnesium ions involved in DNA cleavage and DNA religation<br /> The central core of the protein contains a Toprim fold and a DNA-binding core that contains a winged helix domain (WHD), often referred to as a CAP domain. The catalytic tyrosine lies on this WHD. The DNA-binding core consists of the WHD, which leads to a tower domain. . A coiled-coil region leads to a C-terminal domain that forms the main dimer interface<br /> the Toprim domain is flexible and that this flexibility can allow the Toprim domain to coordinate with the WHD to form a competent cleavage complex<br /> DNA was bent by ~150 degrees through an invariant isoleucine (in topoisomerase II it is I833 and in gyrase it is I172)<br /> The first structure of a C-terminal domain of gyrase was solved by Corbett et al. (Proceedings of the National Academy of Science, 2004, PDB ID = 1SUU)<br /> The structures formed a novel beta barrel, which bends DNA by wrapping the nucleic acid around itself. The bending of DNA by gyrase has been proposed as a key mechanism in the ability of gyrase to introduce negative supercoils into the DNA. This is consistent with footprinting data that shows <br /> that gyrase has a 140-base-pair footprint. Both gyrase and topoisomerase IV CTDs bend DNA, but only gyrase introduces negative supercoils.<br /> <br /> ----<br /> <br /> =='''Reaction'''==<br /> The cleavage of DNA is achieved by a '''transesterification reaction between the tyrosines (Tyr122) and the target phosphoryl groups on opposing strands of the DNA backbone''', resulting in the tyrosine being covalentlty attached to the 5' end of the cleaved segment with a 4-base overhang.<br /> The '''active site tyrosines (Tyr 122)''' are on loops at either end of the dimer interface, 30Å apart, and sit at the ends of strongly basic grooves created by the dimer-related monomers.<br /> <br /> The gyrase structure reveals a '''new cluster of conserved residues''', juxtaposing Tyr 122 and Arg 121 from one monomer and His 80, Arg 32 and Lys 42 from the other monomer. '''This cluster may form the active site of the breakage–reunion reaction''', with the other conserved positive charges (Arg 46 and Arg 47) anchoring the non-covalently bound 3' end of the cleaved DNA.<br /> <Structure load='1ab4_mm1.pdb' size='300' frame='true' align='right' caption='Biological Unit (dimer)' scene='56/568026/Biounit/1' /></div>

Laurent Thomas https://proteopedia.org/index.php?title=File:1ab4_mm1.pdb&diff=1878938 File:1ab4 mm1.pdb 2013-12-24T15:34:46Z

<p>Laurent Thomas: Biological Unit of 1ab4</p> <hr /> <div>== Summary ==<br /> Biological Unit of 1ab4<br /> == Licensing ==<br /> {{PD-self}}</div>

Laurent Thomas https://proteopedia.org/index.php?title=Sandbox_Reserved_828&diff=1878936 Sandbox Reserved 828 2013-12-24T15:31:20Z

<p>Laurent Thomas: </p> <hr /> <div><br /> {{Sandbox_Reserved_ESBS}}<br /> <br /> <br /> ----<br /> <Structure load='1ab4' size='400' frame='true' align='right' caption='Breakage/Reunion domain at 2.8Å' scene='56/568026/Coloured/1'><br /> [[Image:1ab4.png|left|200px]]<br /> '''Introduction :'''<br /> <br /> Gyrase is the '''only prokaryote DNA topoisomerase II able to introduce negative supercoils in the DNA''' in order to remove positive supercoils. It '''catalyses the hydrolysis of two phosphodiester bonds in a DNA segment''' (called G segment). Then, thanks to '''ATP dependant conformation changes''' it enables the passage of another segment (the T segment) through the break, and then religates the broken segment. Gyrase acts prior to the replication (before the replication fork) or other mecanisms requiring loose DNA. <br /> '''In abscence of ATP, like other topoisomerases II, gyrase only relaxes supercoils.'''<br /> <br /> Gyrase is coded by two differents contiguous genes gyrA and GgyrB as it is a 350 kDa A2B2 heterotetramers of two A proteins and two B proteins. <br /> The A protein breaks and religates DNA . The B protein has ATPase activity<br /> <br /> ----<br /> '''Structure'''<br /> [http://jenalib.fli-leibniz.de/ImgLibPDB/thumbnail/manual/1ab4.pdb1_small.gif Biological Unit]<br /> <br /> [[Image:Gyrasegene.jpg]]<br /> <br /> '''The A protein''' breaks and religates DNA as the DNA cleavage core and the CTD lies on it. <br /> The gyrA 59kDa N-ter domain is called Breakage and reunion domain It is composed of two domains at the head region : a winged-helix-turn-helix domain or winged helix domain (WHD) where lies the catalytic tyrosines and a tower domain with alpha/beta structure.<br /> And a single domain with a helical core at the tail region. Two long helices (a14 anda18) emanate from this core and connect, together with the C-terminal helix (a19), the head and tail fragments. <br /> The three connecting helices (a14,a18 anda19) adopt very different conformations, leading to large quaternary movements involving a single hinge-point within the helices and rigid body movements of <br /> the head fragments . <br /> The tail is structurally conserved although large surface loops emanating from different points give it a different outward appearance It forms a heart-shaped homodimer with two protein interfaces, the DNA- and C-gates. GyrA59 is the minimal fragment of the A-subunit which, when complexed with the B-subunit, has DNA-cleavage activity. <br /> The remaining 30–35 kDa comprising the C-terminal domain (CTD) of GyrA shows a domain forming a b-pinwheel with a positively charged amino-acid perimeter. This carboxy-terminal domain of GyrA (cyan) is required for the introduction of DNA supercoils). <br /> <br /> <br /> The B protein has the ATPase domain and the Toprim fold on it. Two ATPase domains dimerize to form a closed conformation. The Toprim fold is a Rossmann fold that contains three invariant acidic residues that coordinate magnesium ions involved in DNA cleavage and DNA religation<br /> The central core of the protein contains a Toprim fold and a DNA-binding core that contains a winged helix domain (WHD), often referred to as a CAP domain. The catalytic tyrosine lies on this WHD. The DNA-binding core consists of the WHD, which leads to a tower domain. . A coiled-coil region leads to a C-terminal domain that forms the main dimer interface<br /> the Toprim domain is flexible and that this flexibility can allow the Toprim domain to coordinate with the WHD to form a competent cleavage complex<br /> DNA was bent by ~150 degrees through an invariant isoleucine (in topoisomerase II it is I833 and in gyrase it is I172)<br /> The first structure of a C-terminal domain of gyrase was solved by Corbett et al. (Proceedings of the National Academy of Science, 2004, PDB ID = 1SUU)<br /> The structures formed a novel beta barrel, which bends DNA by wrapping the nucleic acid around itself. The bending of DNA by gyrase has been proposed as a key mechanism in the ability of gyrase to introduce negative supercoils into the DNA. This is consistent with footprinting data that shows <br /> that gyrase has a 140-base-pair footprint. Both gyrase and topoisomerase IV CTDs bend DNA, but only gyrase introduces negative supercoils.<br /> <br /> ----<br /> <br /> '''Reaction'''<br /> <br /> The cleavage of DNA is achieved by a '''transesterification reaction between the tyrosines (Tyr122) and the target phosphoryl groups on opposing strands of the DNA backbone''', resulting in the tyrosine being covalentlty attached to the 5' end of the cleaved segment with a 4-base overhang.<br /> The '''active site tyrosines (Tyr 122)''' are on loops at either end of the dimer interface, 30Å apart, and sit at the ends of strongly basic grooves created by the dimer-related monomers.<br /> <br /> The gyrase structure reveals a '''new cluster of conserved residues''', juxtaposing Tyr 122 and Arg 121 from one monomer and His 80, Arg 32 and Lys 42 from the other monomer. '''This cluster may form the active site of the breakage–reunion reaction''', with the other conserved positive charges (Arg 46 and Arg 47) anchoring the non-covalently bound 3' end of the cleaved DNA.</div>

Laurent Thomas https://proteopedia.org/index.php?title=Sandbox_Reserved_828&diff=1878920 Sandbox Reserved 828 2013-12-24T14:40:42Z

<p>Laurent Thomas: </p> <hr /> <div><br /> {{Sandbox_Reserved_ESBS}}<br /> <br /> <br /> ----<br /> <Structure load='1ab4' size='400' frame='true' align='right' caption='Breakage/Reunion domain at 2.8Å' scene='Insert optional scene name here' /><br /> [[Image:1ab4.png|left|200px]]<br /> '''Introduction :'''<br /> <br /> Gyrase is the '''only prokaryote DNA topoisomerase II able to introduce negative supercoils in the DNA''' in order to remove positive supercoils. It '''catalyses the hydrolysis of two phosphodiester bonds in a DNA segment''' (called G segment). Then, thanks to '''ATP dependant conformation changes''' it enables the passage of another segment (the T segment) through the break, and then religates the broken segment. Gyrase acts prior to the replication (before the replication fork) or other mecanisms requiring loose DNA. <br /> '''In abscence of ATP, like other topoisomerases II, gyrase only relaxes supercoils.'''<br /> <br /> Gyrase is coded by two differents contiguous genes gyrA and GgyrB as it is a 350 kDa A2B2 heterotetramers of two A proteins and two B proteins. <br /> The A protein breaks and religates DNA . The B protein has ATPase activity<br /> <br /> ----<br /> '''Structure'''<br /> [http://jenalib.fli-leibniz.de/ImgLibPDB/thumbnail/manual/1ab4.pdb1_small.gif Biological Unit]<br /> <br /> The gyrase structure reveals a dimer contact with a grooved concave surface for binding the G segment and a cluster of conserved charged residues surrounding the active-site tyrosines.<br /> The A protein breaks and religates DNA as the DNA cleavage core and the CTD lies on it. '''GyrA59 is the minimal fragment of the A-subunit which, when complexed with the B-subunit, has DNA-cleavage activity.''' The carboxy-terminal domain of GyrA is required for the introduction of DNA supercoils. Each GyrA59 monomer is composed of two domains at the head region: one similar to the DNA-binding domain of the catabolite-activator protein (CAP), including the helix–turn–helix (HTH) motif; and a second domain with α/β structure (the ‘tower’ domain) and a single domain with a helical core at the tail region. Two long helices (α14 and α18) emanate from this core and connect, together with the C-terminal helix (α19), the head and tail fragments The three connecting helices (α14,α18 and α19) adopt very different conformations, leading to large quaternary movements involving a single hinge-point within the helices and rigid body movements of the head fragments (Fig. 2d).. The tail is structurally conserved although large surface loops emanating from different points give it a different outward appearance<br /> <br /> The B protein has the ATPase domain and the Toprim fold on it. Two ATPase domains dimerize to form a closed conformation. The Toprim fold is a Rossmann fold that contains three invariant acidic residues that coordinate magnesium ions involved in DNA cleavage and DNA religation<br /> The central core of the protein contains a Toprim fold and a DNA-binding core that contains a winged helix domain (WHD), often referred to as a CAP domain. The catalytic tyrosine lies on this WHD. The DNA-binding core consists of the WHD, which leads to a tower domain. . A coiled-coil region leads to a C-terminal domain that forms the main dimer interface<br /> the Toprim domain is flexible and that this flexibility can allow the Toprim domain to coordinate with the WHD to form a competent cleavage complex<br /> DNA was bent by ~150 degrees through an invariant isoleucine (in topoisomerase II it is I833 and in gyrase it is I172)<br /> The first structure of a C-terminal domain of gyrase was solved by Corbett et al. (Proceedings of the National Academy of Science, 2004, PDB ID = 1SUU)<br /> The structures formed a novel beta barrel, which bends DNA by wrapping the nucleic acid around itself. The bending of DNA by gyrase has been proposed as a key mechanism in the ability of gyrase to introduce negative supercoils into the DNA. This is consistent with footprinting data that shows <br /> that gyrase has a 140-base-pair footprint. Both gyrase and topoisomerase IV CTDs bend DNA, but only gyrase introduces negative supercoils.<br /> <br /> ----<br /> <br /> '''Reaction'''<br /> <br /> The cleavage of DNA is achieved by a '''transesterification reaction between the tyrosines (Tyr122) and the target phosphoryl groups on opposing strands of the DNA backbone''', resulting in the tyrosine being covalentlty attached to the 5' end of the cleaved segment with a 4-base overhang.<br /> The '''active site tyrosines (Tyr 122)''' are on loops at either end of the dimer interface, 30Å apart, and sit at the ends of strongly basic grooves created by the dimer-related monomers.<br /> <br /> The gyrase structure reveals a '''new cluster of conserved residues''', juxtaposing Tyr 122 and Arg 121 from one monomer and His 80, Arg 32 and Lys 42 from the other monomer. '''This cluster may form the active site of the breakage–reunion reaction''', with the other conserved positive charges (Arg 46 and Arg 47) anchoring the non-covalently bound 3' end of the cleaved DNA.</div>

Laurent Thomas https://proteopedia.org/index.php?title=Sandbox_Reserved_828&diff=1878919 Sandbox Reserved 828 2013-12-24T14:02:32Z

<p>Laurent Thomas: </p> <hr /> <div><br /> {{Sandbox_Reserved_ESBS}}<br /> <br /> <br /> ----<br /> {{STRUCTURE_1ab4| PDB=1ab4 | SCENE= }}<br /> [[Image:1ab4.png|left|200px]]<br /> '''Introduction :'''<br /> <br /> Gyrase is the only prokaryote DNA topoisomerase II able to introduce negative supercoils in the DNA in order to remove positive supercoils. It catalyses the hydrolysis of two phosphodiester bonds in a DNA segment (called G segment). Then, thanks to ATP dependant conformation changes it enables the passage of another segment (the T segment) through the break, and then religates the broken segment. Gyrase acts prior to the replication (before the replication fork) or other mecanisms requiring loose DNA. In abscence of ATP, like other topoisomerases II, gyrase only relaxes supercoils.<br /> <br /> Gyrase is coded by two differents contiguous genes gyrA and GgyrB as it is a 350 kDa A2B2 heterotetramers of two A proteins and two B proteins. <br /> The A protein breaks and religates DNA . The B protein has ATPase activity<br /> <br /> ----<br /> '''Structure'''<br /> [http://jenalib.fli-leibniz.de/ImgLibPDB/thumbnail/manual/1ab4.pdb1_small.gif Biological Unit]<br /> <br /> The gyrase structure reveals a dimer contact with a grooved concave surface for binding the G segment and a cluster of conserved charged residues surrounding the active-site tyrosines.<br /> The A protein breaks and religates DNA as the DNA cleavage core and the CTD lies on it. '''GyrA59 is the minimal fragment of the A-subunit which, when complexed with the B-subunit, has DNA-cleavage activity.''' The carboxy-terminal domain of GyrA is required for the introduction of DNA supercoils. Each GyrA59 monomer is composed of two domains at the head region: one similar to the DNA-binding domain of the catabolite-activator protein (CAP), including the helix–turn–helix (HTH) motif; and a second domain with α/β structure (the ‘tower’ domain) and a single domain with a helical core at the tail region. Two long helices (α14 and α18) emanate from this core and connect, together with the C-terminal helix (α19), the head and tail fragments The three connecting helices (α14,α18 and α19) adopt very different conformations, leading to large quaternary movements involving a single hinge-point within the helices and rigid body movements of the head fragments (Fig. 2d).. The tail is structurally conserved although large surface loops emanating from different points give it a different outward appearance<br /> <br /> The B protein has the ATPase domain and the Toprim fold on it. Two ATPase domains dimerize to form a closed conformation. The Toprim fold is a Rossmann fold that contains three invariant acidic residues that coordinate magnesium ions involved in DNA cleavage and DNA religation<br /> The central core of the protein contains a Toprim fold and a DNA-binding core that contains a winged helix domain (WHD), often referred to as a CAP domain. The catalytic tyrosine lies on this WHD. The DNA-binding core consists of the WHD, which leads to a tower domain. . A coiled-coil region leads to a C-terminal domain that forms the main dimer interface<br /> the Toprim domain is flexible and that this flexibility can allow the Toprim domain to coordinate with the WHD to form a competent cleavage complex<br /> DNA was bent by ~150 degrees through an invariant isoleucine (in topoisomerase II it is I833 and in gyrase it is I172)<br /> The first structure of a C-terminal domain of gyrase was solved by Corbett et al. (Proceedings of the National Academy of Science, 2004, PDB ID = 1SUU)<br /> The structures formed a novel beta barrel, which bends DNA by wrapping the nucleic acid around itself. The bending of DNA by gyrase has been proposed as a key mechanism in the ability of gyrase to introduce negative supercoils into the DNA. This is consistent with footprinting data that shows <br /> that gyrase has a 140-base-pair footprint. Both gyrase and topoisomerase IV CTDs bend DNA, but only gyrase introduces negative supercoils.</div>

Laurent Thomas https://proteopedia.org/index.php?title=Sandbox_Reserved_828&diff=1878916 Sandbox Reserved 828 2013-12-24T12:59:05Z

<p>Laurent Thomas: </p> <hr /> <div><br /> {{Sandbox_Reserved_ESBS}}<br /> <br /> <br /> ----<br /> {{STRUCTURE_1ab4| PDB=1ab4 | SCENE= }}<br /> [[Image:1ab4.png|left|200px]]<br /> '''Introduction :'''<br /> <br /> Gyrase is the only prokaryote DNA topoisomerase II able to introduce negative supercoils in the DNA in order to remove positive supercoils. It catalyses the hydrolysis of two phosphodiester bonds in a DNA segment (called G segment). Then, thanks to ATP dependant conformation changes it enables the passage of another segment (the T segment) through the break, and then religates the broken segment. Gyrase acts prior to the replication (before the replication fork) or other mecanisms requiring loose DNA. In abscence of ATP, like other topoisomerases II, gyrase only relaxes supercoils.<br /> <br /> Gyrase is coded by two differents contiguous genes gyrA and GgyrB as it is a 350 kDa A2B2 heterotetramers of two A proteins and two B proteins. <br /> The A protein breaks and religates DNA . The B protein has ATPase activity<br /> <br /> ----<br /> '''Structure'''<br /> <br /> The gyrase structure reveals a dimer contact with a grooved concave surface for binding the G segment and a cluster of conserved charged residues surrounding the active-site tyrosines.<br /> The A protein breaks and religates DNA as the DNA cleavage core and the CTD lies on it. GyrA59 is the minimal fragment of the A-subunit which, when complexed with the B-subunit, has DNA-cleavage activity. The carboxy-terminal domain of GyrA is required for the introduction of DNA supercoils. Each GyrA59 monomer is composed of two domains at the head region: one similar to the DNA-binding domain of the catabolite-activator protein (CAP), including the helix–turn–helix (HTH) motif; and a second domain with a/bstructure (the ‘tower’ domain) and a single domain with a helical core at the tail region. Two long helices (α14 and α18) emanate from this core and connect, together with the C-terminal helix (α19), the head and tail fragments The three connecting helices (α14,α18 and α19) adopt very different conformations, leading to large quaternary movements involving a single hinge-point within the helices and rigid body movements of the head fragments (Fig. 2d).. The tail is structurally conserved although large surface loops emanating from different points give it a different outward appearance<br /> <br /> The B protein has the ATPase domain and the Toprim fold on it. Two ATPase domains dimerize to form a closed conformation. The Toprim fold is a Rossmann fold that contains three invariant acidic residues that coordinate magnesium ions involved in DNA cleavage and DNA religation<br /> The central core of the protein contains a Toprim fold and a DNA-binding core that contains a winged helix domain (WHD), often referred to as a CAP domain. The catalytic tyrosine lies on this WHD. The DNA-binding core consists of the WHD, which leads to a tower domain. . A coiled-coil region leads to a C-terminal domain that forms the main dimer interface<br /> the Toprim domain is flexible and that this flexibility can allow the Toprim domain to coordinate with the WHD to form a competent cleavage complex<br /> DNA was bent by ~150 degrees through an invariant isoleucine (in topoisomerase II it is I833 and in gyrase it is I172)<br /> The first structure of a C-terminal domain of gyrase was solved by Corbett et al. (Proceedings of the National Academy of Science, 2004, PDB ID = 1SUU)<br /> The structures formed a novel beta barrel, which bends DNA by wrapping the nucleic acid around itself. The bending of DNA by gyrase has been proposed as a key mechanism in the ability of gyrase to introduce negative supercoils into the DNA. This is consistent with footprinting data that shows <br /> that gyrase has a 140-base-pair footprint. Both gyrase and topoisomerase IV CTDs bend DNA, but only gyrase introduces negative supercoils.</div>

Laurent Thomas https://proteopedia.org/index.php?title=Sandbox_Reserved_828&diff=1878914 Sandbox Reserved 828 2013-12-24T12:58:11Z

<p>Laurent Thomas: </p> <hr /> <div><br /> {{Sandbox_Reserved_ESBS}}<br /> <br /> <br /> ----<br /> {{STRUCTURE_1ab4| PDB=1ab4 | SCENE= }}<br /> [[Image:1ab4.png|left|200px]]<br /> '''Introduction :'''<br /> <br /> Gyrase is the only prokaryote DNA topoisomerase II able to introduce negative supercoils in the DNA in order to remove positive supercoils. It catalyses the hydrolysis of two phosphodiester bonds in a DNA segment (called G segment). Then, thanks to ATP dependant conformation changes it enables the passage of another segment (the T segment) through the break, and then religates the broken segment. Gyrase acts prior to the replication (before the replication fork) or other mecanisms requiring loose DNA. In abscence of ATP, like other topoisomerases II, gyrase only relaxes supercoils.<br /> <br /> Gyrase is coded by two differents contiguous genes gyrA and GgyrB as it is a 350 kDa A2B2 heterotetramers of two A proteins and two B proteins. <br /> The A protein breaks and religates DNA . The B protein has ATPase activity<br /> <br /> ----<br /> '''Structure'''<br /> <br /> The gyrase structure reveals a dimer contact with a grooved concave surface for binding the G segment and a cluster of conserved charged residues surrounding the active-site tyrosines.<br /> The A protein breaks and religates DNA as the DNA cleavage core and the CTD lies on it. GyrA59 is the minimal fragment of the A-subunit which, when complexed with the B-subunit, has DNA-cleavage activity. The carboxy-terminal domain of GyrA is required for the introduction of DNA supercoils. Each GyrA59 monomer is composed of two domains at the head region: one similar to the DNA-binding domain of the catabolite-activator protein (CAP), including the helix–turn–helix (HTH) motif; and a second domain with a/bstructure (the ‘tower’ domain) and a single domain with a helical core at the tail region. Two long helices (a14 anda18) emanate from thiscore and connect, together with the C-terminal helix (α19), the head and tail fragments The three connecting helices (α14,α18 and α19) adopt very different conformations, leading to large quaternary movements involving a single hinge-point within the helices and rigid body movements of the head fragments (Fig. 2d).. The tail is structurally conserved although large surface loops emanating from different points give it a different outward appearance<br /> <br /> The B protein has the ATPase domain and the Toprim fold on it. Two ATPase domains dimerize to form a closed conformation. The Toprim fold is a Rossmann fold that contains three invariant acidic residues that coordinate magnesium ions involved in DNA cleavage and DNA religation<br /> The central core of the protein contains a Toprim fold and a DNA-binding core that contains a winged helix domain (WHD), often referred to as a CAP domain. The catalytic tyrosine lies on this WHD. The DNA-binding core consists of the WHD, which leads to a tower domain. . A coiled-coil region leads to a C-terminal domain that forms the main dimer interface<br /> the Toprim domain is flexible and that this flexibility can allow the Toprim domain to coordinate with the WHD to form a competent cleavage complex<br /> DNA was bent by ~150 degrees through an invariant isoleucine (in topoisomerase II it is I833 and in gyrase it is I172)<br /> The first structure of a C-terminal domain of gyrase was solved by Corbett et al. (Proceedings of the National Academy of Science, 2004, PDB ID = 1SUU)<br /> The structures formed a novel beta barrel, which bends DNA by wrapping the nucleic acid around itself. The bending of DNA by gyrase has been proposed as a key mechanism in the ability of gyrase to introduce negative supercoils into the DNA. This is consistent with footprinting data that shows <br /> that gyrase has a 140-base-pair footprint. Both gyrase and topoisomerase IV CTDs bend DNA, but only gyrase introduces negative supercoils.</div>

Laurent Thomas https://proteopedia.org/index.php?title=Sandbox_Reserved_828&diff=1878911 Sandbox Reserved 828 2013-12-24T12:29:52Z

<p>Laurent Thomas: </p> <hr /> <div><br /> {{Sandbox_Reserved_ESBS}}<br /> <br /> <br /> ----<br /> <br /> '''Introduction :'''<br /> <br /> Gyrase is the only prokaryote DNA topoisomerase II able to introduce negative supercoils in the DNA in order to remove positive supercoils. It catalyses the hydrolysis of two phosphodiester bonds in a DNA segment (called G segment). Then, thanks to ATP dependant conformation changes it enables the passage of another segment (the T segment) through the break, and then religates the broken segment. Gyrase acts prior to the replication (before the replication fork) or other mecanisms requiring loose DNA. In abscence of ATP, like other topoisomerases II, gyrase only relaxes supercoils.<br /> <br /> Gyrase is coded by two differents contiguous genes gyrA and GgyrB as it is a 350 kDa A2B2 heterotetramers of two A proteins and two B proteins. <br /> The A protein breaks and religates DNA . The B protein has ATPase activity<br /> <br /> ----<br /> '''Structure'''<br /> <br /> The gyrase structure reveals a dimer contact with a grooved concave surface for binding the G segment and a cluster of conserved charged residues surrounding the active-site tyrosines.<br /> The A protein breaks and religates DNA as the DNA cleavage core and the CTD lies on it. GyrA59 is the minimal fragment of the A-subunit which, when complexed with the B-subunit, has DNA-cleavage activity. The carboxy-terminal domain of GyrA is required for the introduction of DNA supercoils. Each GyrA59 monomer is composed of two domains at the head region: one similar to the DNA-binding domain of the catabolite-activator protein (CAP), including the helix–turn–helix (HTH) motif; and a second domain with a/bstructure (the ‘tower’ domain) and a single domain with a helical core at the tail region. Two long helices (a14 anda18) emanate from thiscore and connect, together with the C-terminal helix (α19), the head and tail fragments The three connecting helices (α14,α18 and α19) adopt very different conformations, leading to large quaternary movements involving a single hinge-point within the helices and rigid body movements of the head fragments (Fig. 2d).. The tail is structurally conserved although large surface loops emanating from different points give it a different outward appearance<br /> <br /> The B protein has the ATPase domain and the Toprim fold on it. Two ATPase domains dimerize to form a closed conformation. The Toprim fold is a Rossmann fold that contains three invariant acidic residues that coordinate magnesium ions involved in DNA cleavage and DNA religation<br /> The central core of the protein contains a Toprim fold and a DNA-binding core that contains a winged helix domain (WHD), often referred to as a CAP domain. The catalytic tyrosine lies on this WHD. The DNA-binding core consists of the WHD, which leads to a tower domain. . A coiled-coil region leads to a C-terminal domain that forms the main dimer interface<br /> the Toprim domain is flexible and that this flexibility can allow the Toprim domain to coordinate with the WHD to form a competent cleavage complex<br /> DNA was bent by ~150 degrees through an invariant isoleucine (in topoisomerase II it is I833 and in gyrase it is I172)<br /> The first structure of a C-terminal domain of gyrase was solved by Corbett et al. (Proceedings of the National Academy of Science, 2004, PDB ID = 1SUU)<br /> The structures formed a novel beta barrel, which bends DNA by wrapping the nucleic acid around itself. The bending of DNA by gyrase has been proposed as a key mechanism in the ability of gyrase to introduce negative supercoils into the DNA. This is consistent with footprinting data that shows <br /> that gyrase has a 140-base-pair footprint. Both gyrase and topoisomerase IV CTDs bend DNA, but only gyrase introduces negative supercoils.</div>

Laurent Thomas https://proteopedia.org/index.php?title=User:Laurent_Thomas/Sandbox_1&diff=1867268 User:Laurent Thomas/Sandbox 1 2013-11-24T18:53:27Z

<p>Laurent Thomas: New page: This is my own sandbox !</p> <hr /> <div>This is my own sandbox !</div>

Laurent Thomas https://proteopedia.org/index.php?title=User:Laurent_Thomas&diff=1867267 User:Laurent Thomas 2013-11-24T18:49:52Z

<p>Laurent Thomas: </p> <hr /> <div>* Full Real Name:Laurent Thomas<br /> <br /> * Position: student<br /> <br /> * Institution (NO ABBREVIATIONS): biotechnologies school of strasbourg, France<br /> <br /> * City, State/Province, Country: Illkirch-graffenstaden, France<br /> <br /> * Field of Expertise or Study: Biotechnologies<br /> <br /> *[[User:Laurent Thomas/Sandbox 1]]</div>

Laurent Thomas