Probing the Two-Gate Mechanism of DNA Gyrase Using Cysteine Cross-Linking

Williams, Nicola L.; Maxwell, Anthony

doi:10.1021/bi9912488

Cited by 71 publications

(55 citation statements)

References 30 publications

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“…1B) and thereby lock the enzyme in the closed clamp conformation around the DNA. Previously, a similar approach involving engineered disulfide bonds has been used successfully to probe the requirements for protein gate opening in the reactions catalyzed by both type IA and type II topoisomerases (9)(10)(11). Here, we show that topo70 2XCys forms a salt-stable complex with DNA under conditions that promote the formation of disulfide bonds and is released from the DNA in the presence of DTT.…”

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confidence: 79%

DNA relaxation by human topoisomerase I occurs in the closed clamp conformation of the protein

Carey

Schultz

Sisson

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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In cocrystal structures of human topoisomerase I and DNA, the enzyme is tightly clamped around the DNA helix. After cleavage and covalent attachment of the enzyme to the 3 end at the nick, DNA relaxation requires rotation of the DNA helix downstream of the cleavage site. Models based on the cocrystal structure reveal that there is insufficient space in the protein for such DNA rotation without some deformation of the cap and linker regions of the enzyme. Alternatively, it is conceivable that the protein clamp opens to facilitate the rotation process. To distinguish between these two possibilities, we engineered two cysteines into the opposing loops of the ''lips'' region of the enzyme, which allowed us to lock the protein via a disulfide crosslink in the closed conformation around the DNA. Importantly, the rate of DNA relaxation when the enzyme was locked on the DNA was comparable to that observed in the absence of the disulfide crosslink. These results indicate that DNA relaxation likely proceeds without extensive opening of the enzyme clamp. D NA topoisomerases are ubiquitous and essential enzymes that solve the topological problems accompanying key nuclear processes such as DNA replication, transcription, repair, and chromatin assembly by introducing temporary single-or double-strand breaks in the DNA (1-4). In addition, these enzymes fine-tune the steady-state level of DNA supercoiling to facilitate protein interactions with DNA and to prevent excessive supercoiling that is deleterious. There are two fundamental types of topoisomerases, which differ in both mechanism and cellular function (1). Type II enzymes are dimeric, promote the passage of one region of duplex DNA through a double-stranded break in the same or a different molecule, and are primarily dedicated to such processes as DNA supercoiling and chromosome segregation. Type I enzymes are monomeric and transiently break one strand of the duplex DNA, allowing for adjustments in helical winding. These enzymes are primarily responsible for removing torsional stress generated by processes that leave the DNA overwound or underwound. The type I topoisomerases have been further divided into two subfamilies based on sequence comparisons and reaction mechanisms (2). The type IA subfamily is characterized by covalent attachment of the enzyme to the 5Ј end of the broken strand in the nicked intermediate, whereas all members of the type IB subfamily attach to the 3Ј end of the broken strand. The prototype of the type IB subfamily, human topoisomerase I, catalyzes changes in the superhelical state of duplex DNA by transiently breaking one strand of the DNA to allow rotation of one region of the duplex relative to another region (5). Strand cleavage is achieved by the nucleophilic attack of the active site tyrosine on a DNA phosphodiester bond. The resulting formation of a phosphodiester bond between the tyrosine and the 3Ј end of the cleaved strand enables the enzyme to reseal the DNA by simple reversal of the cleavage reaction (1).Human topoisomerase I is a 765...

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confidence: 79%

DNA relaxation by human topoisomerase I occurs in the closed clamp conformation of the protein

Carey

Schultz

Sisson

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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“…Structures of fragments of Escherichia coli GyrB (4) and GyrA (5,6), and of a fusion of parts of GyrB and GyrA (7) have been determined, but the overall architecture of gyrase remains unknown. DNA supercoiling by gyrase occurs via a strandpassage mechanism (8)(9)(10)(11). A double-stranded DNA segment binds to the DNA-gate (5,12), close to the catalytic tyrosines that perform the cleavage reaction.…”

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confidence: 99%

DNA-induced narrowing of the gyrase N-gate coordinates T-segment capture and strand passage

Gubaev

Klostermeier

2011

Proc. Natl. Acad. Sci. U.S.A.

147

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DNA gyrase introduces negative supercoils into DNA in an ATPdependent reaction. DNA supercoiling is catalyzed by a strandpassage mechanism, in which a T-segment of DNA is passed through the gap in a transiently cleaved G-segment. Strand passage requires the coordinated closing and opening of three protein interfaces in gyrase, the N-gate, DNA-gate, and C-gate. We show here that DNA binding to the DNA-gate of gyrase and wrapping of DNA around the C-terminal domains of GyrA induces a narrowing of the N-gate. This half-closed state prepares capture of a T-segment in the upper cavity of gyrase. Subsequent N-gate closure upon binding of ATP then poises the reaction toward strand passage. The N-gate reopens after ATP hydrolysis, allowing for further catalytic cycles. DNA binding, cleavage, and wrapping and N-gate narrowing are intimately linked events that coordinate conformational changes at the DNA and the N-gate.gyrase dynamics | nucleotide-induced conformational changes | topoisomerase mechanism | DNA wrapping | negative supercoiling

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“…Structural analysis indicates that the GyrA breakage-reunion domain resembles a clamp with two sets of jaws at opposite ends (18). Experimental studies, using yeast topoisomerase II or DNA gyrase, indicate that DNA is transported by a "two-gate" mechanism, entering the interior of the enzyme through an upper N gate at the GyrA head dimer interface and leaving the enzyme through an "exit gate" at the GyrA bottom dimer interface closer to the C termini (19,20,26).DNA gyrase is the target of two classes of inhibitors, the quinolones, such as nalidixic acid (Nal), which trap DNA gyrase covalently bound to its cleaved substrate, and the coumarins, such as novobiocin (Nov), which inhibit the ATPase activity (15). Mutations conferring Nal resistance are for the most part located in a region of the gyrA gene specifying the N-terminal domain portion between amino acids 51 and 106, the so-called quinolone resistance-determining region (22).…”

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confidence: 99%

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confidence: 99%

“…The crystal structure reveals that the GyrA59 dimer exhibits two regions of interface, the head dimer interface and the bottom dimer interface located at the tails. The role of the bottom interface (the "exit gate") in DNA strand passage has been shown using cysteine cross-linking (26). In the model of the corresponding region of the Salmonella enzyme, the residues T467 and L463 appear to be among the most buried residues at the primary dimer interface.…”

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confidence: 99%

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Mutation at the “Exit Gate” of the Salmonella Gyrase A Subunit Suppresses a Defect in the Gyrase B Subunit

et al. 2005

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In Salmonella enterica serovar Typhimurium, an S431P substitution in the B subunit of gyrase (allele gyrB651) confers resistance to nalidixic acid and causes reduced DNA superhelicity and hypersensitivity to novobiocin. Selection for novobiocin resistance allowed isolation of a mutation in the gyrA gene (allele gyrA659), a T467S substitution, which partially suppresses the supercoiling defect of gyrB651. Modeling analysis suggests that this mutation acts by destabilizing the GyrA bottom dimer interface. This is the first example of a gyrA mutation that compensates for a gyrB defect.DNA gyrase is a member of the family of type II DNA topoisomerases, a group of enzymes that catalyze the interconversion of different topological forms of DNA. DNA gyrase introduces supercoils into DNA through the breakage of a DNA duplex, the passage of another DNA segment through the break, and the resealing of the break (8). This activity involves the opening and closing of a series of molecular gates, coupled to ATP hydrolysis. DNA gyrase is a heterotetramer of two A subunits (97 kDa) and two B subunits (90 kDa), encoded by the gyrA and gyrB genes, respectively (8). The 64-kDa Nterminal domain of GyrA is thought to intervene in the breakage-reunion reaction, whereas the 33-kDa C-terminal domain is involved in the stabilization of the gyrase-DNA complex. The GyrB protein contains a 43-kDa N-terminal ATP binding domain and a 47-kDa C-terminal domain that interacts with GyrA and DNA. Structural analysis indicates that the GyrA breakage-reunion domain resembles a clamp with two sets of jaws at opposite ends (18). Experimental studies, using yeast topoisomerase II or DNA gyrase, indicate that DNA is transported by a "two-gate" mechanism, entering the interior of the enzyme through an upper N gate at the GyrA head dimer interface and leaving the enzyme through an "exit gate" at the GyrA bottom dimer interface closer to the C termini (19,20,26).DNA gyrase is the target of two classes of inhibitors, the quinolones, such as nalidixic acid (Nal), which trap DNA gyrase covalently bound to its cleaved substrate, and the coumarins, such as novobiocin (Nov), which inhibit the ATPase activity (15). Mutations conferring Nal resistance are for the most part located in a region of the gyrA gene specifying the N-terminal domain portion between amino acids 51 and 106, the so-called quinolone resistance-determining region (22). Resistance to quinolones (albeit to a lower level) can also result from mutations in the gyrB gene, and the affected sites identified are amino acids D426, K447, and S464 in the GyrB C-terminal domain (12,27). In addition to their effect on quinolone sensitivity, mutations at sites D426 and K447 also inhibit the gyrase catalytic activity (13). It has been proposed that the quinolone-binding site is a pocket surrounded by surfaces involving the quinolone resistance-determining regions of both the GyrA and GyrB proteins (13,27).Isolation of the gyrB651 allele and its phenotypic suppressor, gyrA659. Mutants of Salmonella enterica ...

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Probing the Two-Gate Mechanism of DNA Gyrase Using Cysteine Cross-Linking

Cited by 71 publications

References 30 publications

DNA relaxation by human topoisomerase I occurs in the closed clamp conformation of the protein

DNA relaxation by human topoisomerase I occurs in the closed clamp conformation of the protein

DNA-induced narrowing of the gyrase N-gate coordinates T-segment capture and strand passage

Mutation at the “Exit Gate” of the Salmonella Gyrase A Subunit Suppresses a Defect in the Gyrase B Subunit

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